Methods of treating s. aureus-associated diseases

ABSTRACT

The present invention provides for methods of preventing and/or treating  S. aureus -associated bacteremia and sepsis, and methods for preventing and/or treating  S. aureus -associated pneumonia in immunocompromised patients using anti- S. aureus  alpha-toxin (anti-AT) antibodies. Also provided are methods of reducing  S. aureus  bacterial load in the bloodstream or heart of a mammalian subject comprising administering to the subject an effective amount of an isolated anti- S. aureus  alpha toxin (anti-AT) antibody or antigen-binding fragment thereof. Methods of reducing  S. aureus  bacterial agglutination and/or thromboembolic lesion formation in a mammalian subject comprising administering to the subject an effective amount of an isolated anti- S. aureus  alpha toxin (anti-AT) antibody or antigen-binding fragment thereof, are also provided. Also provided are methods of preventing or reducing the severity of  S. aureus -associated pneumonia in an immunocompromised mammalian subject.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides for methods of preventing and/or treatingS. aureus-associated bacteremia and sepsis, and methods for preventingand/or treating S. aureus-associated pneumonia in immunocompromisedpatients using anti-S. aureus alpha-toxin (anti-AT) antibodies.

2. Background Art

Staphylococcus aureus (S. aureus) is a leading cause of mortality andmorbidity worldwide, causing a diverse array of infections ranging frommild skin and soft-tissue infections to serious invasive diseases suchendocarditis, osteomyelitis, and necrotizing pneumonia (Lowy F D, N EnglJ Med, 339(8): 520-32 (1998); Klevens et al, JAMA 298(15): 1763-71(2007). S. aureus is commonly classified as either methicillin resistant(MRSA) or methicillin sensitive (MSSA). Several reports have shown thatS. aureus infections result in serious outcomes regardless of resistancestatus (Fowler et al, Arch Intern Med. 163(17):2066-72 (2003); de Krakeret al, PLoS Med. October; 8(10):e1001104 (2011).

Antibiotics are standard of care for treating S. aureus disease. Despitethe introduction of new antibiotics against S. aureus, emergence of newresistance mechanisms requires new approaches to prevent or treat S.aureus diseases. Prior to the antibiotic era, passive administration ofimmune sera to infected patients was used clinically to treat bacterialinfections (Keller and Stiehm, Clin Microbiol Rev 13(4):602-14 (2000)).Today, similar methods are used to treat some toxin-mediated bacterialdiseases (e.g., botulism, diphtheria, tetanus) (Keller and Stiehm, ClinMicrobiol Rev 13(4):602-14 (2000); Arnon et al, N. Engl. J. Med. 354:462-471 (2006). S. aureus alpha toxin (AT) has been shown to be a keyvirulence determinant (amongst numerous other extracellular factors) inseveral S. aureus disease models (e.g., dermonecrosis, pneumonia,sepsis, endocarditis, and mastitis) by comparing S. aureus strainsdeficient for AT expression with isogenic wild-type parent strains(Bramley et al, Infect Immun. 57(8):2489-94 (1989); Bayer et al, Infect.Immun. 65: 4652-4660 (1997); Kernodle et al, Infect. Immun. 65: 179-184(1997); Bubeck Wardenburg et al, Infect Immun. 75(2):1040-4 (2007);Bubeck Wardenburg et al, J Exp Med. 205(2):287-94 (2008); Kobayashi etal, J Infect Dis. 204(6):937-41 (2011)).

AT is a cytolytic 33 kDa pore-forming toxin produced by 90% of S. aureusstrains and is considered to be a major virulence factor. It is secretedas a monomer and binds the specific receptor ADAM-10 on target cellmembranes (Wilke and Bubeck Wardenburg, PNAS 107(30):13473-8 (2010);Inoshima et al, Nat Med 17(10):1310-4 (2011). AT oligomerizes into aheptameric prepore and undergoes a conformational change resulting intransmembrane β-barrel formation and subsequent cell lysis (Bhakdi andTranum-Jensen, 1991; Song et al, 1996). Platelets, along withepithelial, endothelial, and immune cells (e.g., lymphocytes andmacrophages), are susceptible to AT-lysis, suggesting the toxin hasdirect impact on tissue damage and immune evasion (Bhakdi andTranum-Jensen, Microbiol Rev. 55(4):733-51 (1991); Ragle and BubeckWardenburg, Infect Immun. 77(7):2712-8 (2009); Tkaczyk et al, ClinVaccine Immunol 19(3):377-85 (2012)). At sub-lytic concentrations, AThas also been demonstrated to exert significant cytotoxic effects(Grimminger et al, J Immunol. 159(4):1909-16 (1997); Wilke and BubeckWardenburg, PNAS 107(30):13473-8 (2010); Inoshima et al, Nat Med17(10):1310-4 (2011)). AT binding and oligomerization on macrophagemembranes activates the NLRP3 inflammasome that, along with otherstaphylococcal pathogen-associated molecular patterns (PAMPs), inducesIL-1β secretion and promotes cell death (Craven et al, PLoS One 4(10)(2009); Kebaier et al, J Infect Dis 205(5):807-17 (2012). Increasedproinflammatory cytokine expression (e.g. IL-1β) is a hallmark of acutelung injury (Goodman et al, Cytokine Growth Factor Rev. 14(6):523-35(2003)).

AT also activates ADAM-10 mediated proteolysis of E-cadherin present incell-cell adhesive contacts at sub-lytic concentrations, leading to adisruption in epithelium integrity and contributing to epithelial damageseen in pneumonia and skin and soft tissue infections (Inoshima et al,Nat Med 17(10):1310-4 (2011); Maretzky et al, PNAS 102(26):9182-7(2005); Inoshima et al, J Invest Dermatol. 132(5):1513-6 (2012). ATexerts its cytotoxic effects through direct and indirect activities tocreate an environment conducive for bacterial growth and invasivedisease. Consequently, targeted inhibition of AT could prevent or limitS. aureus-associated disease. This hypothesis is supported other studieswhich demonstrate reductions in S. aureus disease severity in murineinfection models following active or passive immunization directedagainst AT (Menzies and Kernodle, Infect Immun 64(5):1839-41(1996);Bubeck Wardenburg et al, J Exp Med. 205(2):287-94 (2008); Ragle andBubeck Wardenburg, Infect Immun 77(7):2712-8 (2009); Kennedy et al, JInfect Dis. 202(7):1050-8 (2010); Tkaczyk et al, Clin Vaccine Immunol19(3):377-85 (2012)).

An anti-AT antibody having an Fc variant region and its parent antibodyLC10 are human, high-affinity, anti-AT mAbs (previously disclosed inU.S. Prov. Appl. No. 61/440,581 and in Intl. Appl. No. PCT/US2012/024201(published as WO2012/109205), the contents of each of which are hereinincorporated by reference) and in Tkaczyk et al., Clinical and VaccineImmunology, 19(3): 377 (2012).

Bacteremia and septic shock account for the majority of Staphylococcusaureus invasive disease (Klevens, et al, JAMA, 298(15): 1763-71 (2007).AT has been proposed to be an important virulence factor during S.aureus sepsis and to be responsible for endothelial damage during sepsis(Powers, et al, J Infect Dis. 206(3):352-6 (2012). Interaction of ATwith its receptor on endothelial cells allows the toxin to mediatevascular damage by direct cell lysis or activation of ADAM-10-mediatedproteolysis of endothelial tight junctions (Id.). Both mechanisms wouldincrease vascular permeability, a hallmark of bacterial sepsis.

While passive immunization with anti-AT monoclonal antibodies has beenshown to result in a significant increase in survival in a murine modelof staphylococcal pneumonia as described in U.S. Prov. Appl. No.61/440,581 and in Intl. Appl. No. PCT/US2012/024201, it is not knownwhether anti-AT antibodies are effective in increasing survival inimmunocompromised mammals having S. aureus associated diseases. This isa critical piece to understand as immunocompromised individuals,particularly those suffering from neutropenia, are at increased risk forS. aureus infections (Andrews and Sullivan, Clin Microbiol Rev.16(4):597-621 (2003); Bouma et al., Br J Haematol. 151(4):312-26(2010)).

The present invention provides, for the first time, a demonstration thatanti-AT antibodies are effective in prophylaxis in sepsis and inimmunocompromised pneumonia.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods for preventing or reducing the severity ofS. aureus-associated sepsis in a mammalian subject comprisingadministering to the subject an effective amount of an isolated anti-S.aureus alpha toxin (anti-AT) antibody or antigen-binding fragmentthereof. Also provided are methods of reducing S. aureus bacterial loadin the bloodstream or heart of a mammalian subject comprisingadministering to the subject an effective amount of an isolated anti-S.aureus alpha toxin (anti-AT) antibody or antigen-binding fragmentthereof. Methods of reducing S. aureus bacterial agglutination and/orthromboembolic lesion formation in a mammalian subject comprisingadministering to the subject an effective amount of an isolated anti-S.aureus alpha toxin (anti-AT) antibody or antigen-binding fragmentthereof, are also provided. Also provided are methods of preventing orreducing the severity of S. aureus-associated pneumonia in animmunocompromised mammalian subject, comprising administering to thesubject an effective amount of an isolated anti-S. aureus alpha toxin(anti-AT) antibody or antigen-binding fragment thereof.

In the various methods described herein, S. aureus bacterial load in thebloodstream or heart of the subject is suitably reduced, and inadditional embodiments, S. aureus bacterial agglutination and/orthromboembolic lesion formation in the subject is reduced.

Suitably, the mammalian subject in the various methods described hereinis human.

In the various methods, the isolated anti-AT antibody or antigen-bindingfragment thereof is selected from the group consisting of Fv, Fab, Fab′,and F(ab′)2. In other embodiments, the antibody is a full-lengthantibody. In still further embodiments, the antibody comprises an Fcvariant region.

In embodiments of the various methods described herein, the isolatedantibody or antigen-binding fragment thereof immunospecifically binds toa Staphylococcus aureus alpha toxin polypeptide and includes:

-   -   (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:        7, 10, 13 or 69;    -   (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:        8, 11, 14, 17, 70 or 75;    -   (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:        9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78;    -   (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1        or 4;    -   (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:        2, 5, 73 or 77; and    -   (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:        3, 6, 64, 68 or 74.

In embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 for use in the various methods described herein correspond to theamino acid sequences of SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10,11, 12, 1, 2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7,17, 18, 1, 2 and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8,65, 1, 2 and 64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8,67, 1, 2 and 68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8,78, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 69, 70,71, 1, 2 and 68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75,71, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69,75, 76, 1, 77 and 74; SEQ ID NOs: 69, 70, 71, 1, 77 and 74

In additional embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a heavy chain variable domain having at least90% identity to the amino acid sequence of SEQ ID NO: 20, 22, 24, 26,28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and (iii)comprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46,48, 50, 52, 54, 56, 58, 60 or 63. Suitably, the isolated antibody orantigen-binding fragment thereof comprises a heavy chain variable domainof SEQ ID NO 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79,59, 61, or 62 and a light chain variable domain of SEQ ID NO: 19, 21,23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.

In additional embodiments of the various methods described herein, theVH and VL correspond to the amino acid sequences of SEQ ID NOs: 20 and19; SEQ ID NOs; 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25;SEQ ID NOs: 28 and 27; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQID NOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 47 and 48; SEQ IDNOs: 49 and 50; SEQ ID NOs: 51 and 52; SEQ ID NOs: 51 and 52; SEQ IDNOs: 53 and 54; SEQ ID NOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ IDNOs: 59 and 60; SEQ ID NOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ IDNOs: 62 and 63; SEQ ID NOs: 79 and 63.

In still further embodiments of the various methods, the isolatedantibody or antigen-binding fragment thereof comprises an anti-ATantibody having an Fc variant domain, wherein the antibody comprises aVH-IgG1-YTE corresponding to SEQ ID NO: 80 and/or a VL-Kappacorresponding to SEQ ID NO: 81.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. LC10 prophylaxis improves survival in an IV lethal challengemodel. Mice (10 per group) were passively immunized with LC10 (45 and 15mg/kg), or an isotype control (R347, 45 mg/kg) 24-h prior to IVchallenge with SF8300 (3×10⁸ cfu). Survival was monitored for 14 days.Data are representative of 4 independent experiments. Statisticalsignificance was assessed with a log rank (Martel-Cox) test:*p-value=0.0005; ** p-value=0.0043).

FIG. 2. LC10 prophylaxis reduces bacterial load in heart. Mice passivelyimmunized with LC10 (45 and 15 mg/kg) or an isotype control (R347, 45mg/kg) 24 hr prior to IV challenge SF8300 (2.98e8 cfu). Fourteen hourspost-infection hearts from infected animals were collected and processedfor CFU enumeration. Statistical analysis was performed with an unpaired2-tailed Student's t-test: *p-value=0.0028; **p-value=0.0082).

FIG. 3. LC10 prophylaxis reduces staphylococcal bacteremia. Mice werepassively immunized with LC10 (45 and 15 mg/kg) or an isotype control(R347, 45 mg/kg) 24 h prior to IV challenge SF8300 (3×10⁸ cfu). Atvarious time points postinfection, blood was collected by cardiacpuncture and plated for CFU enumeration. Statistical analysis wasperformed with a student t test. Data were considered statisticallydifferent vs R347 if *p-value<0.05.

FIG. 4. Total and Differential White Blood Cell Counts. C57BL6/J micewere given 6 different doses of CPM (mg/kg) at Day 0 and Day 3. Bloodsamples of 5 mice per time point per group were taken on Days 0, 1, 4,and 6. Total and differential white blood cell counts (neutrophils,lymphocytes) were analyzed using a Sysmex automated hematology analyzer.

FIG. 5. Bacterial Dose Titration. Five immunocompromised mice were INchallenged with 50 μL log-phased bacterial suspension (dose ranging from1×10⁷ to 2×10⁸ CFU) 24 hours after the second dose of CPM (Day 4).Animal survival was observed for a 7-day period.

FIG. 6. LC10 Increases Survival in Immunocompromised Animals. CPMinjected animals were administered LC10 (45 or 15 mg/kg) or R347 (45mg/kg) 24 hr prior to IN infection with 50 μL of a bacterial suspensionSF8300 (5×10⁷ CFU). Animal survival was monitored for 5 days.Statistical significance was determined using Log-rank test and *indicate statistical difference relative to animals treated with R347(p<0.0001).

FIG. 7. Total and Differential White Blood Cell Counts. C57BL/6 micewere given 2 doses of CPM (150 mg/kg and 100 mg/kg) on Days −4 and −1,respectfully. Blood samples from 5 mice were collected on Days −4, −3,−1, 0, 2, and 3. Total and differential white blood cell counts(neutrophils, lymphocytes) were determined using a Sysmex automatedhematology analyzer.

DETAILED DESCRIPTION OF THE INVENTION

The terms “polypeptide,” “peptide,” “protein,” and “protein fragment”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids.

As used herein, “recombinant” includes reference to a protein producedusing cells that do not have, in their native state, an endogenous copyof the DNA able to express the protein. The cells produce therecombinant protein because they have been genetically altered by theintroduction of the appropriate isolated nucleic acid sequence.

As used herein, “antibody” and “immunoglobulin” are used interchangeablyin the broadest sense and include monoclonal antibodies (e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andantigen-binding fragments, as described herein. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Each heavy chain has at one end a variable domain(V_(H)) followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend. The terms “constant” and “variable” are used functionally.

The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light andheavy chain variable domains (Clothia, et al., J. Mol. Biol. 186, 651-66(1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596(1985)). Five human immunoglobulin classes are defined on the basis oftheir heavy chain composition, and are named IgG, IgM, IgA, IgE, andIgD. The IgG-class and IgA-class antibodies are further divided intosubclasses, namely, IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2. Theheavy chains in IgG, IgA, and IgD antibodies have three constant regiondomains, that are designated CH1, CH2, and CH3, and the heavy chains inIgM and IgE antibodies have four constant region domains, CH1, CH2, CH3,and CH4. Thus, heavy chains have one variable region and three or fourconstant regions.

Immunoglobulin structure and function are reviewed, for example, inHarlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14, ColdSpring Harbor Laboratory, Cold Spring Harbor (1988).

References to “V_(H)” or a “VH” refer to the variable region of animmunoglobulin heavy chain, including an Fv, scFv, dsFv or Fab.

References to “V_(L)” or a “VL” refer to the variable region of animmunoglobulin light chain, including an Fv, scFv, dsFv, or Fab.

The term “antigen-binding fragment” refers to a portion of an intactantibody and refers to the antigenic determining variable regions of anintact antibody. Examples of antigen-binding fragments include, but arenot limited to Fab, Fab′, F(ab′)2, Fv and single chain Fv fragments,linear antibodies, single chain antibodies, and multispecific antibodiesformed from antigen-binding fragments.

The terms “single chain Fv” or “scFv” refers to an antibody in which thevariable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain. Termsinclude binding molecules which consist of one light chain variabledomain (V_(L)) or portion thereof, and one heavy chain variable domain(VH) or portion thereof, wherein each variable domain (or portionthereof) is derived from the same or different antibodies. scFvmolecules typically comprise an scFv linker interposed between the V_(H)domain and the V_(L) domain. scFv molecules are known in the art and aredescribed, _(e.g.), in U.S. Pat. No. 5,892,019, Ho, et al., Gene77:51-59 (1989); Bird, et al., Science 242:423-426 (1988); Pantoliano,et al., Biochemistry 30:10117-10125 (1991); Milenic, et al., CancerResearch 51:6363-6371 (1991); Takkinen, et al., Protein Engineering4:837-841 (1991), all of which are hereby incorporated by reference intheir entireties.

Anti-S. aureus Alpha Toxin Antibodies and Antigen-Binding Fragments

An anti-S. aureus alpha toxin (also referred to as anti-S. aureus AT oranti-AT) antibody or antigen-binding fragment thereof, as utilizedherein, immunospecifically binds to one or more epitopes specific to thealpha toxin protein, peptide, subunit, fragment, portion, oligomers orany combination thereof and generally do not specifically bind to otherpolypeptides. The term “oligomers” or “alpha toxin oligomers” refers toan association of alpha toxin monomers (e.g., 2 monomers, 3 monomers, 4monomers, 5 monomers, 6 monomers or 7 monomers) to form a functionalpore (e.g., 7 alpha toxin monomers). An epitope can comprise at leastone antibody binding region that comprises at least one portion of thealpha toxin protein. The term “epitope” as used herein refers to aprotein determinant capable of binding to an antibody. Epitopesgenerally include chemically active surface groupings of molecules suchas amino acids and/or sugar side chains and generally have specificthree dimensional structural characteristics, as well as specificchemical characteristics (e.g., charge, polarity, basic, acidic,hydrophobicity and the like). Conformational and non-conformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. In someembodiments, the epitope recognized interferes with formation of theactive heptamer (e.g., inhibits oligomerization of alpha toxin monomersinto an active heptamer complex).

In certain embodiments, an epitope is comprised of at least a portion ofthe alpha toxin protein, which is involved in formation of an alphatoxin heptamer complex. A specified epitope can comprise any combinationof at least one amino acid sequence of at least 3 amino acid residues tothe entire specified portion of contiguous amino acids of the alphatoxin protein. In some embodiments, the epitope is at least 4 amino acidresidues, at least 5 amino acid residues, at least 6 amino acidresidues, at least 7 amino acid residues, at least 8 amino acidresidues, at least 9 amino acid residues, at least 10 amino acidresidues, at least 11 amino acid residues, at least 12 amino acidresidues, at least 13 amino acid residues, at least 14 amino acidresidues, or at least 15 amino acid residues to the entire specifiedportion of contiguous amino acids of the alpha toxin protein. In certainother embodiments, the epitope comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or 15 contiguous or non-contiguous amino acid residues.In further embodiments, the amino acid residues comprised within theepitope are involved in alpha toxin heptamer complex formation.

Thus, in specific embodiments, isolated/purified anti-alpha toxinantibodies and antigen-binding fragments immunospecifically bind to amolecule comprising the amino acid sequence according to SEQ ID NO: 39and/or to a molecule comprising the amino acid sequence according to SEQID NO: 40. In certain embodiments, anti-alpha toxin antibodies andantigen-binding fragments also bind alpha toxin homologs or orthologsfrom different species, or to variants of the amino acid sequence of SEQID NO: 39, where the histidine at position 35 is replaced with leucine,or replaced with other amino acids corresponding to H35 mutations knownto one of ordinary skill in the art.

Variable Regions

In certain embodiments, an anti-alpha toxin antibody or antigen-bindingfragment is prepared from a parent antibody. In some embodiments, theanti-alpha toxin antibody or antigen-binding fragment is encompassedwithin the parent antibody. As used herein, the term “parent antibody”refers to an antibody that is encoded by an amino acid sequence used forthe preparation of the variant or derivative, defined herein. A parentpolypeptide may comprise a native antibody sequence (i.e., a naturallyoccurring, including a naturally occurring allelic variant) or anantibody sequence with pre-existing amino acid sequence modifications(such as other insertions, deletions and/or substitutions) of anaturally occurring sequence. A parent antibody may be a humanizedantibody or a human antibody. In specific embodiments, anti-alpha toxinantibodies and antigen-binding fragments are variants of the parentantibody. As used herein, the term “variant” refers to an anti-alphatoxin antibody or antigen-binding fragment that differs in amino acidsequence from a “parent” anti-alpha toxin antibody or antigen-bindingfragment amino acid sequence by virtue of addition, deletion and/orsubstitution of one or more amino acid residue(s) in the parent antibodysequence.

The antigen-binding portion of an antibody comprises one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., alpha toxin). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody (i.e., antigen-binding fragments). Examples of“antigen-binding fragments” encompassed within the “antigen-bindingportion” of an antibody include (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment, whichconsists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Although the two domains of the Fv fragment,VL and VH, often are coded for by separate genes, they can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the VL and VH regions pair toform monovalent molecules (known as single chain Fv (scFv)). Such singlechain antibodies also are encompassed within the terms “antibody and“antigen-binding fragment” of an antibody. These antigen-bindingfragments can be obtained using known techniques, and the fragments canbe screened for binding activity in the same manner as are intactantibodies. Antigen-binding fragments can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins.

The present anti-alpha toxin antibodies and antigen-binding fragmentscomprise at least one antigen binding domain. In some embodiments, ananti-alpha toxin antibody or antigen-binding fragment comprises a VHcomprising the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41,43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In certainembodiments, an anti-alpha toxin antibody or antigen-binding fragmentcomprises a VL comprising the amino acid sequence of SEQ ID NO: 19, 21,23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In yet anotherembodiment, an anti-alpha toxin antibody or antigen-binding fragmentcomprises a VH comprising the amino acid sequence of SEQ ID NO: 20, 22,24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and aVL comprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27,42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. See Table 7 for arepresentation of VH and VL sequences as presented herein which can bepresent in any combination to form an anti-alpha toxin antibody orantigen-binding fragment, or present in a combination to form a mAb ofthe invention. In some embodiments, the VH is selected from SEQ ID NO:20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or62. In various embodiments, the VL is selected from SEQ ID NO: 19, 21,23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. Certain VH andVL nucleotide sequences are presented in Table 8.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises a VH and a VL, where the VH and VL have amino acidsequences represented by SEQ ID NOs: 20 and 19; SEQ ID NOs; 22 and 21;SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27; SEQID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ IDNOs: 47 and 48; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; SEQ IDNOs: 51 and 52; SEQ ID NOs: 51 and 52; SEQ ID NOs: 53 and 54; SEQ IDNOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ ID NOs: 59 and 60; SEQ IDNOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ ID NOs: 62 and 63; SEQ IDNOs: 79 and 63.

Tables 1-7 provide heavy chain variable regions (VH), light chainvariable regions (VL), and complementarity determining regions (CDRs)for certain embodiments of the antibodies and antigen-binding fragmentspresented herein. In certain embodiments, anti-alpha toxin antibodiesand antigen-binding fragments comprise a VH and/or VL that has a givenpercent identify to at least one of the VH and/or VL sequences disclosedin Table 7. As used herein, the term “percent (%) sequence identity”,also including “homology” is defined as the percentage of amino acidresidues or nucleotides in a candidate sequence that are identical withthe amino acid residues or nucleotides in the reference sequences, suchas parent antibody sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Optimal alignment of the sequences for comparisonmay be produced, besides manually, by means of local homology algorithmsknown in the art or by means of computer programs which use thesealgorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

In specific embodiments an antibody or antigen-binding fragmentimmunospecifically binds to alpha toxin and comprises a heavy chainvariable domain comprising at least 90% identity to the amino acidsequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53,55, 57, 79, 59, 61, or 62 and comprises a light chain variable domaincomprising at least 90% identity to the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63,where the antibody has the activity of inhibiting the binding of one ormore alpha toxin monomers to each other (e.g., inhibitsoligomerization).

Complementarity Determining Regions

While the variable domain (VH and VL) comprises the antigen-bindingregion, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in segments calledComplementarity Determining Regions (CDRs), both in the light chain (VLor VK) and the heavy chain (VH) variable domains. The more highlyconserved portions of the variable domains are called the frameworkregions (FR). The variable domains of native heavy and light chains eachcomprise four FR, largely adopting a β-sheet configuration, connected bythree CDRs, which form loops connecting, and in some cases forming partof, the beta-sheet structure. The CDRs in each chain are held togetherin close proximity by the FR and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies(see, Kabat et al., supra). The three CDRs of the heavy chain aredesignated VH-CDR1, VH CDR2, and VH-CDR-3, and the three CDRs of thelight chain are designated VL-CDR1, VL-CDR2, and Vl-CDR3. The Kabatnumbering system is used herein. As such, VH-CDR1 begins atapproximately amino acid 31 (i.e., approximately 9 residues after thefirst cysteine residue), includes approximately 5-7 amino acids, andends at the next serine residue. VH-CDR2 begins at the fifteenth residueafter the end of CDR-H1, includes approximately 16-19 amino acids, andends at the next glycine residue. VH-CDR3 begins at approximately thethirtieth amino acid residue after the end of VH-CDR2; includesapproximately 13-15 amino acids; and ends at the sequence M-D-V. VL-CDR1begins at approximately residue 24 (i.e., following a cysteine residue);includes approximately 10-15 residues; and ends with the sequence Y-V-S.VL-CDR2 begins at approximately the sixteenth residue after the end ofVL-CDR1 and includes approximately 7 residues. VL-CDR3 begins atapproximately the thirty-third residue after the end of VH-CDR2;includes approximately 7-11 residues and ends at the sequence T-I-L.Note that CDRs vary considerably from antibody to antibody (and bydefinition will not exhibit homology with the Kabat consensussequences).

The present anti-alpha toxin antibodies and antigen-binding fragmentscomprise at least one antigen binding domain that includes at least onecomplementarity determining region (CDR1, CDR2 or CDR3). In someembodiments, an anti-alpha toxin antibody or antigen-binding fragmentcomprises a VH that includes at least one VH CDR (e.g., CDR-H1, CDR-H2or CDR-H3). In certain embodiments, an anti-alpha toxin antibody orantigen-binding fragment comprises a VL that includes at least one VLCDR (e.g., CDR-L1, CDR-L2 or CDR-L3).

In some embodiments, the isolated antibody or antigen-binding fragmentthereof that immunospecifically binds to a Staphylococcus aureus alphatoxin polypeptide includes, (a) a VH CDR1 comprising an amino acidsequence identical to, or comprising 1, 2, or 3 amino acid residuesubstitutions relative to SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2comprising an amino acid sequence identical to, or comprising 1, 2, or 3amino acid residue substitutions relative to SEQ ID NO: 8, 11, 14, 17,70 or 75; and (c) a VH CDR3 comprising an amino acid sequence identicalto, or comprising 1, 2, or 3 amino acid residue substitutions relativeto SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78.

In particular embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a VH CDR1, VH CDR2 and VH CDR3 comprisingamino acid sequences identical to, or comprising 1, 2, or 3 amino acidresidue substitutions in each CDR relative to SEQ ID NOs: 7, 8 and 9;SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 7, 17and 18; SEQ ID NOs: 7, 8 and 16; SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7,8 and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs:69, 70 and 71; SEQ ID NOs: 7, 8 and 72; SEQ ID NOs: 69, 75 and 71; SEQID NOs: 69, 75 and 76; or SEQ ID NOs: 69, 70 and 71.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof that immunospecifically binds to a Staphylococcus aureus alphatoxin polypeptide includes, (a) a VL CDR1 comprising an amino acidsequence identical to, or comprising 1, 2, or 3 amino acid residuesubstitutions relative to SEQ ID NO: 1 or 4; (b) a VL CDR2 comprising anamino acid sequence identical to, or comprising 1, 2, or 3 amino acidresidue substitutions relative to SEQ ID NO: 2, 5, 73 or 77; and (c) aVL CDR3 comprising an amino acid sequence identical to, or comprising 1,2, or 3 amino acid residue substitutions relative to SEQ ID NO: 3, 6,64, 68 or 74.

In particular embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a VL CDR1, VL CDR2 and VL CDR3 comprisingamino acid sequences identical to, or comprising 1, 2, or 3 amino acidresidue substitutions in each CDR relative to SEQ ID NOs: 1, 2 and 3;SEQ ID NOs: 4, 5 and 6; SEQ ID NOs: 1, 2 and 64; SEQ ID NOs: 1, 2 and68; SEQ ID NOs: 1, 73 and 74; or SEQ ID NOs: 1, 77 and 74.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof that immunospecifically binds to a Staphylococcus aureus alphatoxin polypeptide comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3 comprising amino acid sequences identical to, orcomprising 1, 2, or 3 amino acid residue substitutions in each CDRrelative to: (a) a VH CDR1 comprising the amino acid sequence of SEQ IDNO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence ofSEQ ID NO: 8, 11, 14, 17, 70 or 75; (c) a VH CDR3 comprising the aminoacid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5,73, or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQID NO: 3, 6, 64, 68 or 74.

In particular embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 comprising amino acid sequences identical to, orcomprising 1, 2, or 3 amino acid residue substitutions in each CDRrelative to SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1,2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 69, 70, 71, 1, 2 and68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77and 74; SEQ ID NOs: 69, 70, 71, 1, 77 and 74,

In some embodiments, provided is a composition that comprises anisolated antibody or antigen-binding fragment thereof that (i) includesa VH chain domain comprising three CDRs and a VL chain domain comprisingthree CDRs; and (ii) immunospecifically binds to a Staphylococcus aureusalpha toxin polypeptide, where the three CDRs of the VH chain domaininclude (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising the aminoacid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or78. In particular embodiments, the VH CDR1, VH CDR2 and VH CDR3correspond to SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ IDNOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16;SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ IDNOs: 69, 70 and 71.

Also provided in certain embodiments is a composition that comprises anisolated antibody or antigen-binding fragment thereof that (i) includesa VH chain domain comprising three CDRs and a VL chain domain comprisingthree CDRs; and (ii) immunospecifically binds to a Staphylococcus aureusalpha toxin polypeptide, where the three CDRs of the VL chain domaininclude (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1or 4; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2,5, 73, or 77; and (c) a VL CDR3 comprising the amino acid sequence ofSEQ ID NO: 3, 6, 64, 68 or 74. In particular embodiments, the VL CDR1,VL CDR2 and VL CDR3 correspond to SEQ ID NOs: 1, 2 and 3; SEQ ID NOs: 4,5 and 6; SEQ ID NOs: 1, 2 and 64; SEQ ID NOs: 1, 2 and 68; SEQ ID NOs:1, 73 and 74; or SEQ ID NOs: 1, 77 and 74.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof that immunospecifically binds to a Staphylococcus aureus alphatoxin polypeptide comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3 comprising amino acid sequences identical to, orcomprising 1, 2, or 3 amino acid residue substitutions in each CDRrelative to: (a) a VH CDR1 comprising the amino acid sequence of SEQ IDNO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence ofSEQ ID NO: 8, 11, 14, 17, 70 or 75; (c) a VH CDR3 comprising the aminoacid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5,73, or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQID NO: 3, 6, 64, 68 or 74.

In particular embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 comprising amino acid sequences identical to, orcomprising 1, 2, or 3 amino acid residue substitutions in each CDRrelative to SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1,2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 69, 70, 71, 1, 2 and68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77and 74; SEQ ID NOs: 69, 70, 71, 1, 77 and 74,

In some embodiments, provided is a composition that comprises anisolated antibody or antigen-binding fragment thereof that (i) includesa VH chain domain comprising three CDRs and a VL chain domain comprisingthree CDRs; and (ii) immunospecifically binds to a Staphylococcus aureusalpha toxin polypeptide, where the three CDRs of the VH chain domaininclude (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising the aminoacid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or78. In particular embodiments, the VH CDR1, VH CDR2 and VH CDR3correspond to SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ IDNOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16;SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ IDNOs: 69, 70 and 71.

Provided also in some embodiments are compositions that include anisolated antibody or antigen-binding fragment thereof that (i)immunospecifically binds to a Staphylococcus aureus alpha toxinpolypeptide, (ii) comprises a heavy chain variable domain comprising atleast 90% identity to the amino acid sequence of SEQ ID NO: 20, 22, 24,26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and (iii)comprises a light chain variable domain comprising at least 90% identityto the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46,48, 50, 52, 54, 56, 58, 60 or 63.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof includes a heavy chain variable domain of SEQ ID NO: 20, 22, 24,26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and alight chain variable domain of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44,46, 48, 50, 52, 54, 56, 58, 60 or 63.

In particular embodiments, the isolated antibody or antigen-bindingfragment thereof comprises a VH and a VL, where the VH and VL are eachidentical to or each have at least 90%, 95% or 98% identity to the VHand VL amino acid sequences of SEQ ID NOs: 20 and 19; SEQ ID NOs; 22 and21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27;SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQID NOs: 47 and 48; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; SEQ IDNOs: 51 and 52; SEQ ID NOs: 51 and 52; SEQ ID NOs: 53 and 54; SEQ IDNOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ ID NOs: 59 and 60; SEQ IDNOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ ID NOs: 62 and 63; SEQ IDNOs: 79 and 63.

Variant Fc Regions

The present invention also includes binding members of the invention,and in particular the antibodies of the invention, that have modifiedIgG constant domains. Antibodies of the human IgG class, which havefunctional characteristics such as long half-life in serum and theability to mediate various effector functions are used in certainembodiments of the invention (Monoclonal Antibodies: Principles andApplications, Wiley-Liss, Inc., Chapter 1 (1995)). The human IgG classantibody is further classified into the following 4 subclasses: IgG1,IgG2, IgG3 and IgG4. A large number of studies have so far beenconducted for ADCC and CDC as effector functions of the IgG classantibody, and it has been reported that among antibodies of the humanIgG class, the IgG1 subclass has the highest ADCC activity and CDCactivity in humans (Chemical Immunology, 65, 88 (1997)).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which non-specific cytotoxic cells (e.g.,Natural Killer (NK) cells, neutrophils, and macrophages) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. In one embodiment, such cells are human cells. While not wishingto be limited to any particular mechanism of action, these cytotoxiccells that mediate ADCC generally express Fc receptors (FcRs). Theprimary cells for mediating ADCC, NK cells, express FcγRIII, whereasmonocytes express FcγRI, FcγRII, FcγRIII and/or FcγRIV. FcR expressionon hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev.Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule, an invitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecules of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA),95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to initiate complement activation and lyse a target in thepresence of complement. The complement activation pathway is initiatedby the binding of the first component of the complement system (Clq) toa molecule (e.g., an antibody) complexed with a cognate antigen. Toassess complement activation, a CDC assay, e.g., as described inGazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

Expression of ADCC activity and CDC activity of the human IgG1 subclassantibodies generally involves binding of the Fc region of the antibodyto a receptor for an antibody (hereinafter referred to as “FcγR”)existing on the surface of effector cells such as killer cells, naturalkiller cells or activated macrophages. Various complement components canbe bound. Regarding the binding, it has been suggested that severalamino acid residues in the hinge region and the second domain of Cregion (hereinafter referred to as “Cγ2 domain”) of the antibody areimportant (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319(1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain inthe Cγ2 domain (Chemical Immunology, 65, 88 (1997)) is also important.

“Effector cells” are leukocytes that express one or more FcRs andperform effector functions. The cells express at least FcγRI, FCγRII,FcγRIII and/or FcγRIV and carry out ADCC effector function. Examples ofhuman leukocytes which mediate ADCC include peripheral blood mononuclearcells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cellsand neutrophils.

The terms “Fe receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment, the FcR is anative sequence human FcR. Moreover, in certain embodiments, the FcR isone that binds an IgG antibody (a gamma receptor) and includes receptorsof the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelicvariants and alternatively spliced forms of these receptors. FcγRIIreceptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an“inhibiting receptor”), which have similar amino acid sequences thatdiffer primarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (See, Daëron, Annu. Rev. Immunol., 15:203-234(1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol.,9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol.,24:249 (1994)).

In certain embodiments, an anti-alpha toxin antibody or antigen-bindingfragment comprises an altered Fc region (also referred to herein as“variant Fc region”) in which one or more alterations have been made inthe Fc region in order to change functional and/or pharmacokineticproperties of the antibodies. Such alterations may result in a decreaseor increase of Clq binding and complement dependent cytotoxicity (CDC)or of FcgammaR binding, for IgG. The present technology encompasses theantibodies described herein with variant Fc regions where changes havebeen made to alter the effector function, providing a desired effect.Accordingly, in some embodiments an anti-alpha toxin antibody orantigen-binding fragment comprises a variant Fc region (i.e., Fc regionsthat have been altered as discussed below). Anti-alpha toxin antibodiesand antigen-binding fragments herein comprising a variant Fc region arealso referred to here as “Fe variant antibodies.” As used herein nativerefers to the unmodified parental sequence and the antibody comprising anative Fc region is herein referred to as a “native Fc antibody”. Insome embodiments, the variant Fc region exhibits a similar level ofinducing effector function as compared to the native Fc region. Incertain embodiments, the variant Fc region exhibits a higher inductionof effector function as compared to the native Fc. In certainembodiments, the variant Fc region exhibits lower induction of effectorfunction as compared to the native Fc. Some specific embodiments ofvariant Fc regions are detailed herein. Methods for measuring effectorfunction are known in the art.

Effector function of an antibody can be modified through changes in theFc region, including but not limited to, amino acid substitutions, aminoacid additions, amino acid deletions and changes in post translationalmodifications to Fc amino acids (e.g., glycosylation). Methods describedbelow may be used to alter the effector function of an isolated antibodyor antigen-binding fragment as described herein, resulting in anantibody or antigen-binding fragment having certain propertiesadvantageous for prophylaxis or treatment of a particular Staphylococcalaureus-associated disease or condition.

In some embodiments an Fc variant antibody is prepared that has alteredbinding properties for an Fc ligand (e.g., an Fc receptor, Clq) relativeto a native Fc antibody. Examples of binding properties include but arenot limited to, binding specificity, equilibrium dissociation constant(K_(d)), dissociation and association rates (koff and kon respectively),binding affinity and/or avidity. It is known in the art that theequilibrium dissociation constant (K_(d)) is defined as koff/kon. Incertain aspects, an antibody comprising an Fc variant region with a lowK_(d) may be more desirable to an antibody with a high K_(d). However,in some instances the value of the kon or koff may be more relevant thanthe value of the K_(d). It can be determined which kinetic parameter ismore important for a given antibody application.

In some embodiments, Fc variant antibodies exhibit altered bindingaffinity for one or more Fc receptors including, but not limited toFcRn, FcgammaRI (CD64) including isoforms FcgammaRIA, FcgammaRIB, andFcgammaRIC; FcgammaRII (CD32 including isoforms FcgammaRIIA,FcgammaRIIB, and FcgammaRIIC); and FcgammaRIII (CD16, including isoformsFcgammaRIIIA and FcgammaRIIIB) as compared to an native Fc antibody.

In certain embodiments, an Fc variant antibody has enhanced binding toone or more Fc ligand relative to a native Fc antibody. In certainembodiments, the Fc variant antibody exhibits increased or decreasedaffinity for an Fc ligand that is at least 2 fold, or at least 3 fold,or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold,or at least 60 fold, or at least 70 fold, or at least 80 fold, or atleast 90 fold, or at least 100 fold, or at least 200 fold, or is between2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 foldand 100 fold, or between 75 fold and 200 fold, or between 100 and 200fold, more or less than a native Fc antibody. In various embodiments, Fcvariant antibodies exhibit affinities for an Fc ligand that are at least90%, at least 80%, at least 70%, at least 60%, at least 50%, at least40%, at least 30%, at least 20%, at least 10%, or at least 5% more orless than an native Fc antibody. In certain embodiments, an Fc variantantibody has increased affinity for an Fc ligand. An Fc variant antibodymay sometimes have decreased affinity for an Fc ligand.

In some embodiments, an Fc variant antibody has enhanced binding to theFc receptor FcgammaRIIIA In some embodiments, an Fc variant antibody hasenhanced binding to the Fc receptor FcgammaRIIB. In certain embodiments,an Fc variant antibody has enhanced binding to both the Fc receptorsFcgammaRIIIA and FcgammaRIIB. In certain embodiments, Fc variantantibodies that have enhanced binding to FcgammaRIIIA do not have aconcomitant increase in binding the FcgammaRIIB receptor as compared toa native Fc antibody. In certain embodiments, an Fc variant antibody hasreduced binding to the Fc receptor FcgammaRIIIA An Fc variant antibodymay sometimes have reduced binding to the Fc receptor FcgammaRIIB. Invarious embodiments, an Fc variant antibody exhibiting altered affinityfor FcgammaRIIIA and/or FcgammaRIIB has enhanced binding to the Fcreceptor FcRn. In some embodiments, an Fc variant antibody exhibitingaltered affinity for FcgammaRIIIA and/or FcgammaRIIB has altered bindingto Clq relative to a native Fc antibody.

In certain embodiments, Fc variant antibodies exhibit affinities forFcgammaRIIIA receptor that are at least 2 fold, or at least 3 fold, orat least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, orat least 60 fold, or at least 70 fold, or at least 80 fold, or at least90 fold, or at least 100 fold, or at least 200 fold, or are between 2fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold,more or less than an native Fc antibody. In various embodiments, Fcvariant antibodies exhibit affinities for FcgammaRIIIA that are at least90%, at least 80%, at least 70%, at least 60%, at least 50%, at least40%, at least 30%, at least 20%, at least 10%, or at least 5% more orless than an native Fc antibody.

In certain embodiments, Fc variant antibodies exhibit affinities forFcgammaRIIB receptor that are at least 2 fold, or at least 3 fold, or atleast 5 fold, or at least 7 fold, or a least 10 fold, or at least 20fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, orat least 60 fold, or at least 70 fold, or at least 80 fold, or at least90 fold, or at least 100 fold, or at least 200 fold, or are between 2fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold,more or less than an native Fc antibody. In certain embodiments, Fcvariant antibodies exhibit affinities for FcgammaRIIB that are at least90%, at least 80%, at least 70%, at least 60%, at least 50%, at least40%, at least 30%, at least 20%, at least 10%, or at least 5% more orless than an native Fc antibody.

In some embodiments, Fc variant antibodies exhibit increased ordecreased affinities to Clq relative to a native Fc antibody. In someembodiments, Fc variant antibodies exhibit affinities for Clq receptorthat are at least 2 fold, or at least 3 fold, or at least 5 fold, or atleast 7 fold, or a least 10 fold, or at least 20 fold, or at least 30fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, orat least 70 fold, or at least 80 fold, or at least 90 fold, or at least100 fold, or at least 200 fold, or are between 2 fold and 10 fold, orbetween 5 fold and 50 fold, or between 25 fold and 100 fold, or between75 fold and 200 fold, or between 100 and 200 fold, more or less than annative Fc antibody. In certain embodiments, Fc variant antibodiesexhibit affinities for Clq that are at least 90%, at least 80%, at least70%, at least 60%, at least 50%, at least 40%, at least 30%, at least20%, at least 10%, or at least 5% more or less than an native Fcantibody. In various embodiments, an Fc variant antibody exhibitingaltered affinity for Ciq has enhanced binding to the Fc receptor FcRn.In yet another specific embodiment, an Fc variant antibody exhibitingaltered affinity for Clq has altered binding to FcgammaRIIIA and/orFcgammaRIIB relative to a native Fc antibody.

It is contemplated that Fc variant antibodies are characterized by invitro functional assays for determining one or more FcgammaR mediatedeffector cell functions. In certain embodiments, Fc variant antibodieshave similar binding properties and effector cell functions in in vivomodels (such as those described and disclosed herein) as those in invitro based assays. The present technology does not exclude Fc variantantibodies that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. The term“antibody half-life” as used herein means a pharmacokinetic property ofan antibody that is a measure of the mean survival time of antibodymolecules following their administration. Antibody half-life can beexpressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body (or other mammal) ora specific compartment thereof, for example, as measured in serum, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

An increase in half-life allows for the reduction in amount of druggiven to a patient as well as reducing the frequency of administration.An increase in half-life can also be beneficial, for example, forpreventing a Staphylococcal aureus-associated disease or condition, andalso for preventing a recurrence of infection that can often occur oncea patient has been released from the hospital. To increase the serumhalf life of the antibody, one may incorporate a salvage receptorbinding epitope into the antibody (especially an antigen-bindingfragment) as known in the art. As used herein, the term “salvagereceptor binding epitope” refers to an epitope of the Fc region of anIgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible forincreasing the in vivo serum half-life of the IgG molecule. Antibodieswith increased half-lives may also be generated by modifying amino acidresidues identified as involved in the interaction between the Fc andthe FcRn receptor. In addition, the half-life of an anti-alpha toxinantibody or antigen-binding fragment may be increased by conjugation toPEG or Albumin by techniques widely utilized in the art. In someembodiments antibodies comprising Fc variant regions of an anti-alphatoxin antibody have an increased half-life of about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about90%, about 95%, about 100%, about 125%, about 150% or more as comparedto an antibody comprising a native Fc region. In some embodimentsantibodies comprising Fc variant regions have an increased half-life ofabout 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold,about 20 fold, about 50 fold or more, or is between 2 fold and 10 fold,or between 5 fold and 25 fold, or between 15 fold and 50 fold, ascompared to an antibody comprising a native Fc region.

In some embodiments, the technology presented herein provides Fcvariants, where the Fc region comprises a modification (e.g., amino acidsubstitutions, amino acid insertions, amino acid deletions) at one ormore positions selected from the group consisting of 234, 235, 236, 237,238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262,263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298,299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443 asnumbered by the EU index as set forth in Kabat. Optionally, the Fcregion may comprise a non-naturally occurring amino acid residue atadditional and/or alternative positions known in the art.

In a certain embodiments, provided herein is an Fc variant, where the Fcregion comprises at least one substitution selected from the groupconsisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V,234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y,235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y,2401, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R. 243W, 243L 243Y,243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E,256M, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W,264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V,265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H,269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N,296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T,298F, 2991, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D,325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N,327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H,328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I,330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E,331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S,332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N,378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EUindex as set forth in Kabat. Optionally, the Fc region may compriseadditional and/or alternative non-naturally occurring amino acidresidues known in the art.

In various embodiments, provided herein is an Fc variant antibody, wherethe Fc region comprises at least one modification (e.g., amino acidsubstitutions, amino acid insertions, amino acid deletions) at one ormore positions selected from the group consisting of 234, 235 and 331.In some embodiments, the non-naturally occurring amino acids areselected from the group consisting of 234F, 235F, 235Y, and 331S .Provided herein is an Fc variant, where the Fc region comprises at leastone non-naturally occurring amino acid at one or more positions selectedfrom the group consisting of 239, 330 and 332. Some embodiments, thenon-naturally occurring amino acids are selected from the groupconsisting of 239D, 330L and 332E.

In some embodiments, provided herein is an Fc variant antibody, wherethe Fc region comprises at least one non-naturally occurring amino acidat one or more positions selected from the group consisting of 252, 254,and 256. In certain embodiments, the non-naturally occurring amino acidsare selected from the group consisting of 252Y, 254T and 256E, describedin U.S. Pat. No. 7,083,784, the contents of which are hereinincorporated by reference in its entirety.

In certain embodiments the effector functions elicited by IgG antibodiesstrongly depend on the carbohydrate moiety linked to the Fc region ofthe protein. Thus, glycosylation of the Fc region can be modified toincrease or decrease effector function. Accordingly, in some embodimentsthe Fc regions of anti-alpha toxin antibodies and antigen-bindingfragments provided herein comprise altered glycosylation of amino acidresidues. In certain embodiments, the altered glycosylation of the aminoacid residues results in lowered effector function. In certainembodiments, the altered glycosylation of the amino acid residuesresults in increased effector function. In some embodiments, the Fcregion has reduced fucosylation. In certain embodiments, the Fc regionis afucosylated.

In some embodiments, the Fc variants herein may be combined with otherknown Fc variants as known in the art. Other modifications and/orsubstitutions and/or additions and/or deletions of the Fc domain can beintroduced. In particular embodiments, an anti-AT antibody of theinvention having an Fc variant domain comprises a VH-IgG1-YTEcorresponding to SEQ ID NO: 80 and/or a VL-Kappa corresponding to SEQ IDNO: 81.

Representative Sequences for Anti-S. aureus at Antibodies

TABLE 1 VL CDR sequences for mAbs 2A3.1, 10A7.5, 12B8.19 and 25E9.1SEQ ID NO: Description Sequence SEQ ID NO: 1 VL CDR1 RASQSISSWLASEQ ID NO: 2 VL CDR2 KASSLES SEQ ID NO: 3 VL CDR3 QQYNSYWT

TABLE 2 VL CDR sequences for mAB 28F6.1 SEQ ID NO: Description SequenceSEQ ID NO: 4 mAb 28F6.1 VL CDR1 RASQGIRNDLG SEQ ID NO: 5mAb 28F6.1 VL CDR2 DASSLQS SEQ ID NO: 6 mAb 28F6.1 VL CDR3 LQDYNYPWT

TABLE 3 VH CDR sequences for mAb 2A3.1 SEQ ID NO: Description SequenceSEQ ID NO: 7 VH CDR1 SYDMH SEQ ID NO: 8 VH CDR2 GIGTAGDTYYPGSVKGSEQ ID NO: 9 VH CDR3 DNYSSTGGYYGMDV

TABLE 4 VH CDR sequences for mAbs 10A7.5 and 12B8.19 SEQ ID NO:Description Sequence SEQ ID NO: 10 VH CDR1 RYDMH SEQ ID NO: 11 VH CDR2VIGTDGDTYYPGSVKG SEQ ID NO: 12 VH CDR3 DRYSSSNHYNGMDV

TABLE 5 VH CDR sequences for mAb 28F6.1 SEQ ID NO: Description SequenceSEQ ID NO: 13 mAb 28F6.1 VH CDR1 SYAMT SEQ ID NO: 14 mAb 28F6.1 VH CDR2VISGSGGSTYYADSVKG SEQ ID NO: 15 mAb 28F6.1 VH CDR3 DGRQVEDYYYYYGMDV

TABLE 6 VH CDR sequences for mAb 25E9.1 SEQ ID NO: Description SequenceSEQ ID NO: 7 mAb 25E9.1 VH CDR1 SYDMH SEQ ID NO: 17 mAb 25E9.1 VH CDR2VIDTAGDTYYPGSVKG SEQ ID NO: 18 mAb 25E9.1 VH CDR3 DRYSGNFHYNGMDV

TABLE 7 VL and VH amino acid sequences for anti-alpha toxin mAbsVH or VL sequence  Description (with CDRs in bold) CDR1 CDR2 CDR3mAb 2A3.1 DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES QQYNSYW VLCRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) T  PKLLIYKASSLESGVPSRFSGSG(SEQ ID NO: 1) (SEQ ID NO: 3) SGTEFTLTISSLQPDDFATYYCQ QYNSYWTFGQGTKVEIK(SEQ ID NO: 19) mAb 2A3.1 EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DNYSSTGGVH AASGFTFSSYDMHWVRQATG (SEQ ID NO: 7) YYPGSVKG YYGMDVKGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8) (SEQ ID NO: 9)VKGRFTISRENAKNSLYLQLNS LRAGDTAVYFCARDNYSSTGG YYGMDVWGQGTTVTVSS(SEQ ID NO: 20) mAb 10A7.5 DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLESQQYNSYW VL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) TPKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1) (SEQ ID NO: 3)SGTEFTLTISSLQPDDFATYYCQ QYNSYWTFGQGTKVEIK (SEQ ID NO: 21) mAb 10A7.5EVQLVESGGGLVQPGGSLRLSC RYDMH VIGTDGDT DRYSSSNH VH AASGFTFSRYDMHWVRQATG(SEQ ID NO: 10) YYPGSVKG YNGMDV KGLEWVSVIGTDGDTYYPGSV (SEQ ID NO: 11)(SEQ ID NO: 12) KGRFIISRENAKNSLYLEMNSL RAGDTAVYYCARDRYSSSNHYNGMDVWGQGTTVTVSS (SEQ ID NO: 22) mAb 12B8.19 DIQMTQSPSTLSASVGDRVTITRASQSISS KASSLES QQYNSYW VL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) TPKVLIYKASSLESGVPSRFSGS (SEQ ID NO: 1) (SEQ ID NO: 3)GSGTEFTLTISSLQPDDFATYYC QQYNSYWTFGQGTKVEIK (SEQ ID NO: 23) mAb 12B8.19EVQLVESGGGLVQPGGSLRLSC RYDMH VIGTDGDT DRYSSSNH VH AASGFTFSRYDMHWVRQATG(SEQ ID NO: 10) YYPGSVKG YNGMDV KGLEWVSVIGTDGDTYYPGSV (SEQ ID NO: 11)(SEQ ID NO: 12) KGRFIISRENAKNSLYLEMNSL RAGDTAVYYCARDRYSSSNHYNGMDVWGQGTTVTVSS (SEQ ID NO: 24) mAb 28F6.1 AIQMTQSPSSLSASVGDRVTITCRASQGIRN DASSLQS LQDYNYP VL RASQGIRNDLGWYQQKPGKA DLG  (SEQ ID NO: 5) WTPKLLIYDASSLQSGVPSRFSGSG (SEQ ID NO: 4) (SEQ ID NO: 6)SGTDFTLTISSLQPEDFATYYCL QDYNYPWTFGQGTKVEIK (SEQ ID NO: 25) mAb 28F6.1EVQLLESGGGLVQPGGSLRLSC SYAMT VISGSGGST DGRQVED VH AASGFTFSSYAMTWVRQAPGK(SEQ ID NO: 13) YYADSVK YYYYYGM GLEWVSVISGSGGSTYYADSV G DVKGRFTVSRDNSKNTLYLQMNS (SEQ ID NO: 14) (SEQ ID NO: 15)LRAEDTAVYYCAKDGRQVED YYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 26) mAb 25E9.1DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES QQYNSYW VL CRASQSISSWLAWYQQKPGKAWLA (SEQ ID NO: 2) T PKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1)(SEQ ID NO: 3) SGTEFTLTISSLQPDDFATYYCQ QYNSYWTFGQGTKVEIK (SEQ ID NO: 27)mAb 25E9.1 EVQLVESGGGLVQPGGSLRLSC SYDMH SVIDTAGD DRYSGNFH VHTASGFTFSSYDMHWVRQATGK (SEQ ID NO: 7) TYYPGSVK YNGMDVGLEWVSVIDTAGDTYYPGSVK G (SEQ ID NO: 18) GRFTISRENAKNSLYLQMNSLR(SEQ ID NO: 17) AGDTAVYYCVRDRYSGNFHY NGMDVWGQGTTVTVSS (SEQ ID NO: 28)mAb QD20 EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DRYSPTGH VHAASGFTFSSYDMHWVRQATG (SEQ ID NO: 7) YYPGSVKG YMGMDV KGLEWVSGIGTAGDTYYPGS(SEQ ID NO: 8) (SEQ ID NO: 16) VKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARDRYSPTG HYMGMDVWGQGTTVTVSS (SEQ ID NO: 41) mAb QD20DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES QQYDTYW VL CRASQSISSWLAWYQQKPGKAWLA (SEQ ID NO: 2) T PKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1)(SEQ ID NO: 64) SGTEFTLTISSLQPDDFATYYCQ QYDTYWTFGQGTKVEIK(SEQ ID NO: 42) mAb QD33 EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DRYSRTGHVH AASGFTFSSYDMHWVRQATG (SEQ ID NO: 7) YYPGSVKG YMGMDVKGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8) (SEQ ID NO: 65)VKGRFTISRENAKNSLYLQMN SLRAGDTAVYYCARDRYSRTG HYMGMDVWGQGTTVTVSS(SEQ ID NO: 43) mAb QD33 DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES QQYDTYWVL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) T PKLLIYKASSLESGVPSRFSGSG(SEQ ID NO: 1) (SEQ ID NO: 64) SGTEFTLTISSLQPDDFATYYCQ QYDTYWTFGQGTKVEIK(SEQ ID NO: 44) mAb QD37 EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DRYSRTGHVH AASGFTFSSYDMHWVRQATG (SEQ ID NO: 7) YYPGSVKG YMGMSLKGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8) (SEQ ID NO: 66)VKGRFTISRENAKNSLYLQMN SLRAGDTAVYYCARDRYSRTG HYMGMSLWGQGTTVTVSS(SEQ ID NO: 45) mAb QD37 DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES QQYDTYWVL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) T PKLLIYKASSLESGVPSRFSGSG(SEQ ID NO: 1) (SEQ ID NO: 64) SGTEFTLTISSLQPDDFATYYCQ QYDTYWTFGQGTKVEIK(SEQ ID NO: 46) mAb QD3 VH EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDTDNYSRTGH AASGFTFSSYDMHWVRQATG (SEQ ID NO: 7) YYPGSVKG YMGMDVKGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8) (SEQ ID NO: 67)VKGRFTISRENAKNSLYLQMN SLRAGDTAVYYCARDNYSRTG HYMGMDVWGQGTTVTVSS(SEQ ID NO: 47) mAb QD3 VL DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLESKQYADYW CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) TPKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1) (SEQ ID NO: 68)SGTEFTLTISSLQPDDFATYYCK QYADYWTFGQGTKVEIK (SEQ ID NO: 48) mAb QD4 VHEVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DNYSRTGH AASGFTFSSYDMHWVRQATG(SEQ ID NO: 7) YYPGSVKG YMGMDV KGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8)(SEQ ID NO: 67) VKGRFTISRENAKNSLYLQMN SLRAGDTAVYYCARDNYSRTGHYMGMDVWGQGTTVTVSS (SEQ ID NO: 49) mAb QD4 VL DIQMTQSPSTLSASVGDRVTITRASQSISS KASSLES QQYDTYW CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) TPKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1) (SEQ ID NO: 64)SGTEFTLTISSLQPDDFATYYCQ QYDTYWTFGQGTKVEIK (SEQ ID NO: 50) mAb QD23EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DRYSPTGH VH AASGFTFSSYDMHWVRQATG(SEQ ID NO: 7) YYPGSVKG YMGMSL KGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8)(SEQ ID NO: 78) VKGRFTISRENAKNSLYLQMN SLRAGDTAVYYCARDRYSPTGHYMGMSLWGQGTTVTVSS (SEQ ID NO: 51) mAb QD23 DIQMTQSPSTLSASVGDRVTITRASQSISS KASSLES QQYDTYW VL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) TPKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1) (SEQ ID NO: 64)SGTEFTLTISSLQPDDFATYYCQ QYDTYWTFGQGTKVEIK (SEQ ID NO: 52) mAb QD32EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DRYSRTGH VH AASGFTFSSYDMHWVRQATG(SEQ ID NO: 7) YYPGSVKG YMGMDV KGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8)NO: 65) VKGRFTISRENAKNSLYLQMN SLRAGDTAVYYCARDRYSRTG HYMGMDVWGQGTTVTVSS(SEQ ID NO: 53) mAb QD32 DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES KQYADYWVL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) T PKLLIYKASSLESGVPSRFSGSG(SEQ ID NO: 1) (SEQ ID NO: 68) SGTEFTLTISSLQPDDFATYYCK QYADYWTFGQGTKVEIK(SEQ ID NO: 54) mAb 2A3GL EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DNYSSTGGVH AASGFTFSSYDMHWVRQATG (SEQ ID NO: 7) YYPGSVKG YYGMDVKGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8) (SEQ ID NO: 9) VKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARDNYSSTG GYYGMDVWGQGTTVTVSS (SEQ ID NO: 55) mAb 2A3GLDIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES QQYNSYW VL CRASQSISSWLAWYQQKPGKAWLA (SEQ ID NO: 2) T PKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1)(SEQ ID NO: 3) SGTEFTLTISSLQPDDFATYYCQ QYNSYWTFGQGTKVEIK (SEQ ID NO: 56)mAb LC10 EVQLVESGGGLVQPGGSLRLSC SHDMH GIGTAGDT DRYSPTGH VHAASGFTFSSHDMHWVRQATG (SEQ ID NO: 69) YYPDSVKG YYGMDVKGLEWVSGIGTAGDTYYPDSV (SEQ ID NO: 70) (SEQ ID NO: 71)KGRFTISRENAKNSLYLQMNSL RAGDTAVYYCARDRYSPTGH YYGMDVWGQGTTVTVSS(SEQ ID NO: 57) mAb LC10 DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES KQYADYWVL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) T PKLLIYKASSLESGVPSRFSGSG(SEQ ID NO: 1) (SEQ ID NO: 68) SGTEFTLTISSLQPDDFATYYCK QYADYWTFGQGTKVEIK(SEQ ID NO: 58) mAb TVES EVQLVESGGGLVQPGGSLRLSC SYDMH GIGTAGDT DNYSPTGGVH AASGFTFSSYDMHWVRQATG (SEQ ID NO: 7) YYPGSVKG YYGMDVKGLEWVSGIGTAGDTYYPGS (SEQ ID NO: 8) (SEQ ID NO: 72)VKGRFTISRENAKNSLYLQMN SLRAGDTAVYYCARDNYSPTG GYYGMDVWGQGTTVTVSS(SEQ ID NO: 59) mAb TVES DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLKS QQYESYWVL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 73) T PKLLIYKASSLKSGVPSRFSGS(SEQ ID NO: 1) (SEQ ID NO: 74) GSGTEFTLTISSLQPDDFATYYCQQYESYWTFGQGTKVEIK (SEQ ID NO: 60) mAb EVQLVESGGGLVQPGGSLRLSC SHDMHGIGTRGDT DRYSPTGH 3H7KAD VH AASGFTFSSHDMHWVRQATG (SEQ ID NO: 69)YYPDSVKG YYGMDV KGLEWVSGIGTRGDTYYPDSV (SEQ ID NO: 75) (SEQ ID NO: 71)KGRFTISRENAKNSLYLQMNSL RAGDTAVYYCARDRYSPTGH YYGMDVWGQGTTVTVSS(SEQ ID NO: 61) mAb DIQMTQSPSTLSASVGDRVTIT RASQSISS KASSLES KQYADYW3H7KAD VL CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) TPKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1) (SEQ ID NO: 68)SGTEFTLTISSLQPDDFATYYCK QYADYWTFGQGTKVEIK (SEQ ID NO: 58) mAb LC9 VHEVQLVESGGGLVQPGGSLRLSC SHDMH GIGTRGDT DKYSPTGH AASGFTFSSHDMHWVRQATG(SEQ ID NO: 69) YYPDSVKG YYGMDV KGLEWVSGIGTRGDTYYPDSV (SEQ ID NO: 75)(SEQ ID NO: 76) KGRFTISRENAKNSLYLQMNSL RAGDTAVYYCARDKYSPTGHYYGMDVWGQGTTVTVSS (SEQ ID NO: 62) mAb LC9 VL DIQMTQSPSTLSASVGDRVTITRASQSISS KASSLES KQYADYW CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 2) TPKLLIYKASSLESGVPSRFSGSG (SEQ ID NO: 1) (SEQ ID NO: 68)SGTEFTLTISSLQPDDFATYYCK QYADYWTFGQGTKVEIK (SEQ ID NO: 58) mAb LC4 VHEVQLVESGGGLVQPGGSLRLSC SHDMH GIGTRGDT DKYSPTGH AASGFTFSSHDMHWVRQATG(SEQ ID NO: 69) YYPDSVKG YYGMDV KGLEWVSG IGTRGDTYYPDSV (SEQ ID NO: 75)(SEQ ID NO: 76) KGRFTISRENAKNSLYLQMNSL RAGDTAVYYCARDKYSPTGHYYGMDVWGQGTTVTVSS (SEQ ID NO: 62) mAb LC4 VL DIQMTQSPSTLSASVGDRVTITRASQSISS KASSLVK QQYESYW CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 77) TPKLLIYKASSLVKGVPSRFSGS (SEQ ID NO: 1) (SEQ ID NO: 74)GSGTEFTLTISSLQPDDFATYYC QQYESYWTFGQGTKVEIK (SEQ ID NO: 63) mAb LC5 VHEVQLVESGGGLVQPGGSLRLSC SHDMH GIGTAGDT DRYSPTGH AASGFTFSSHDMHWVRQATG(SEQ ID NO: 69) YYPDSVKG YYGMDV KGLEWVSGIGTAGDTYYPDSV (SEQ ID NO: 70)(SEQ ID NO: 71) KGRFTISRENAKNSLYLQMNSL RAGDTAVYYCARDRYSPTGHYYGMDVWGQGTTVTVSS (SEQ ID NO: 79) mAb LC5 VL DIQMTQSPSTLSASVGDRVTITRASQSISS KASSLVK QQYESYW CRASQSISSWLAWYQQKPGKA WLA (SEQ ID NO: 77) TPKLLIYKASSLVKGVPSRFSGS (SEQ ID NO: 1) (SEQ ID NO: 74)GSGTEFTLTISSLQPDDFATYYC QQYESYWTFGQGTKVEIK (SEQ ID NO: 63)

TABLE 8 VL and VH nucleotide sequences for anti-alpha toxin mAbsSEQ ID NO: Description Sequence SEQ ID NO: 29 mAb 2A3.1 VL GACATCCAGATGACCCAGTCTCCTTCCAC nucleotide CCTGTCTGCATCTGTAGGAGACAGAGTCAsequence CCATCACTTGCCGGGCCAGTCAGAGTATT AGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCT ATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG GACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACT GCCAACAGTATAATAGTTATTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 30 mAb 2A3.1 VH GAGGTGCAGCTGGTGGAGTCTGGGGGAG nucleotide GCTTGGTACAGCCTGGGGGGTCCCTGAGAsequence CTCTCCTGTGCAGCCTCTGGATTCACCTTC AGTAGCTACGACATGCACTGGGTCCGCCAAGCTACAGGAAAAGGTCTGGAGTGGGTC TCAGGTATTGGCACTGCTGGTGACACATATTATCCAGGCTCCGTGAAGGGCCGATTCA CCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAATTGAACAGCCTGAGAGC CGGGGACACGGCTGTGTACTTCTGTGCAAGAGACAATTATAGCAGCACCGGGGGGTA CTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 31 mAb 10A7.5 VLGACATCCAGATGACCCAGTCTCCTTCCAC nucleotide  CCTGTCTGCATCTGTAGGAGACAGAGTCAsequence CCATCACTTGCCGGGCCAGTCAGAGTATT AGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCT ATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG GACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACT GCCAACAGTATAATAGTTATTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 32 mAb 10A7.5 VHGAGGTGCAGCTGGTGGAGTCTGGGGGAG nucleotide  GCTTGGTACAGCCTGGGGGGTCCCTGAGAsequence CTCTCCTGTGCAGCCTCTGGATTCACCTTC AGTAGGTACGACATGCACTGGGTCCGCCAAGCTACAGGAAAAGGTCTGGAGTGGGTC TCAGTTATTGGTACTGATGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCA TCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTGAAATGAACAGCCTGAGAGC CGGGGACACGGCTGTGTATTACTGTGCAAGAGATCGGTATAGCAGCTCGAACCACTAC AACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 33 mAb 12B8.19 VLGACATCCAGATGACCCAGTCTCCTTCCAC nucleotide  CCTGTCTGCATCTGTAGGAGACAGAGTCAsequence CCATCACTTGCCGGGCCAGTCAGAGTATT AGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATCT ATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG GACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACT GCCAACAGTATAATAGTTATTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 34 mAb 12B8.19 VHGAGGTGCAGCTGGTGGAGTCTGGGGGAG nucleotide  GCTTGGTACAGCCTGGGGGGTCCCTGAGAsequence CTCTCCTGTGCAGCCTCTGGATTCACCTTC AGTAGGTACGACATGCACTGGGTCCGCCAAGCTACAGGAAAAGGTCTGGAGTGGGTC TCAGTTATTGGTACTGATGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCA TCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTGAAATGAACAGCCTGAGAGC CGGGGACACGGCTGTGTATTACTGTGCAAGAGATCGGTATAGCAGCTCGAACCACTAC AACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 35 mAb 28F6.1 VLGCCATCCAGATGACCCAGTCTCCATCCTC nucleotide  CCTGTCTGCATCTGTAGGAGACAGAGTCAsequence CCATCACTTGCCGGGCAAGTCAGGGCATT AGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT ATGATGCATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG CACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTAC TGTCTACAAGATTACAATTACCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATC AAA SEQ ID NO: 36 mAb 28F6.1 VHGAGGTGCAGCTGTTGGAGTCTGGGGGAG nucleotide  GCTTGGTACAGCCTGGGGGGTCCCTGAGAsequence CTCTCCTGTGCAGCCTCTGGATTCACCTTT AGCAGCTATGCCATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTC TCAGTTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGT TCACCGTCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAG AGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGGAGGCAGGTCGAGGATTA CTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 37 mAb 25E9.1 VLGACATCCAGATGACCCAGTCTCCTTCCAC nucleotide  CCTGTCTGCATCTGTAGGAGACAGAGTCAsequence CCATCACTTGCCGGGCCAGTCAGAGTATT AGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCT ATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG GACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACT GCCAACAGTATAATAGTTATTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 38 mAb 25E9.1 VHGAGGTGCAGCTGGTGGAGTCTGGGGGAG nucleotide  GCTTGGTACAGCCTGGGGGGTCCCTGAGAsequence CTCTCCTGTACAGCCTCTGGATTCACCTTC AGTAGTTACGACATGCACTGGGTCCGCCAAGCTACAGGAAAAGGTCTGGAGTGGGTC TCAGTTATTGATACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCA CCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGC CGGGGACACGGCTGTGTATTACTGTGTAAGAGATAGGTATAGTGGGAACTTCCACTAC AACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

TABLE 9 Alpha Toxin VL and VH CDR summary table Description SEQ ID NOsVL CDR 1 1, 4 VL CDR 2 2, 5, 73, 77 VL CDR 3 3, 6, 64, 68, 74 VH CDR 17, 10, 13, 69 VH CDR 2 8, 11, 14, 17, 70, 75 VH CDR 3 9, 12, 15, 18, 16,65, 66, 67, 71, 72, 76, 78

TABLE 10 Alpha Toxin Amino Acid Sequences Staphylococcusadsdiniktgttdigsnttvktgdlvtydken aureus gmhldcvfysfiddknhnldcllvirtkgtiaalpha toxin gqyrvyseeganksglawpsafkvq1q1pdnevaqisdyyprnsidtkeymstltygthgnvtg ddtgkiggliganvsightlkyvqpdfktilesptdldcvgwkvifnnmvnqnwgpydrdswnp vygnqlfmktrngsmkaadnfldpnkassllssgfspdfatvitmdrkaskqqtnidviyervr ddyqlhwtstnwkgtntkdkwtdrsserykidwekeemtn (SEQ ID NO: 39) S. aureus alphaadsdiniktgttdigsnttvktgdlvtydken toxin gmlldcvfysfiddknhnldcllvirtkgtiaH35L mutant gqyrvyseeganksglawpsafkvqlqlpdnevaqisdyyprnsidtkeymstltygthgnvtg ddtgkiggliganvsightlkyvqpdfktilesptdldcvgwkvifnnmvnqnwgpydrdswnp vygnqlfmktrngsmkaadnfldpnkassllssgfspdfatvitmdrkaskqqtnidviyervr ddyqlhwtstnwkgtntkdkwtdrsserykidwekeemtn (SEQ ID NO: 40)

TABLE 11 VL and VH amino acid sequences for anti-alpha toxin mAbshaving Fc variant region SEQ ID NO: Description Sequence SEQ ID NO: 80LC 10-VH-IgG1-YTE: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDMHWVRQATGKGLEWVSGIGTA GDTYYPDSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARDRYSPTGHYY GMDVWGQGTTVTVSS- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO: 81 LC10 VL-KappaDIQMTQSPSTLSASVGDRVTITCRASQSI SSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFAT YYCKQYADYWTFGQGTKVEIK-RTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGE

Pharmaceutical Formulations Comprising Anti-AT Antibodies andAntigen-Binding Fragments Thereof

Also provided are pharmaceutical formulations comprising an anti-alphatoxin antibody or antigen-binding fragment thereof as described hereinand a carrier. Such formulations can be readily administered in thevarious methods described throughout. In some embodiments, theformulation comprises a pharmaceutically acceptable carrier.

As used herein, the pharmaceutical formulations comprising an anti-alphatoxin antibody or antigen-binding fragment thereof are referred to asformulations of the technology. The term “pharmaceutically acceptablecarrier” means one or more non-toxic materials that do not interferewith the effectiveness of the biological activity of the activeingredients. Such preparations may routinely contain salts, bufferingagents, preservatives, compatible carriers, and optionally othertherapeutic agents. Such pharmaceutically acceptable preparations mayalso routinely contain compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration into ahuman. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co mingled with the antibodiesand antigen-binding fragments described herein, and with each other, ina manner such that there is no interaction which would substantiallyimpair the desired pharmaceutical efficacy.

Pharmaceutical compositions as described herein may be formulated for aparticular dosage. Dosage regimens may be adjusted to provide theoptimum desired response. For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound (i.e. antibody or antigen-binding fragment) calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms are dictated by and directly dependent on (a) the uniquecharacteristics of the anti-alpha toxin antibody or antigen-bindingfragment and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an anti-alphatoxin antibody or antigen-binding fragment for the treatment ofsensitivity in individuals.

Therapeutic compositions of the present technology can be formulated forparticular routes of administration, such as oral, nasal, pulmonary,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods knownin the art of pharmacy. The amount of active ingredient (i.e., antibodyor antigen-binding fragment) which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect.

Treatment of S. aureus-Associated Diseases

The present invention also provides methods of preventing and/ortreating S. aureus-associated diseases and conditions, including forexample bacteremia and sepsis using anti-S. aureus alpha-toxin (anti-AT)antibodies and antigen-binding fragments thereof. Also provided aremethods for preventing and/or treating S. aureus-associated diseases andconditions, including for example, pneumonia in immunocompromisedpatients using anti-S. aureus alpha-toxin (anti-AT) antibodies andantigen-binding fragments thereof.

Any of the anti-AT antibodies or antigen-binding fragments thereofdescribed throughout, as well as mutants, variants and derivatives ofsuch antibodies, can be utilized in the various methods describedherein. While exemplary anti-AT antibodies and antigen-binding fragmentsthereof are described herein for use in the various methods and in theExamples provided, it should be understood that any anti-AT antibody orantigen-binding fragment thereof known in the art, and particularlythose described herein and disclosed in Published International PatentApplication No. WO 2012/109285, the disclosure of which is incorporatedby reference herein in its entirety, can be utilized in the variousmethods.

Also known as blood poisoning, bacteremia occurs when S. aureus bacteriaenter a mammal's bloodstream, including humans. A persistent fever isone sign of bacteremia. The bacteria can travel to locations deep withinthe body, to produce infections affecting internal organs, such asbrain, heart, lungs, bones and muscles, or surgically implanted devices,such as artificial joints or cardiac pacemakers. One hallmark of S.aureus sepsis is bacterial agglutination and thromboembolic lesionformation which is measured as bacterial colony forming units (CFU) inthe heart (McAdow et al, 2011).

In embodiments, methods are provided for preventing S. aureus-associatedsepsis in a mammalian subject or reducing the severity of S.aureus-associated sepsis in a mammalian subject. Such methods suitablycomprise administering to the subject an effective amount of an isolatedanti-S. aureus alpha toxin (anti-AT) antibody or antigen-bindingfragment thereof, including isolated anti-S. aureus alpha toxin(anti-AT) antibodies or antigen-binding fragments described herein orotherwise known in the art.

Methods of preventing S. aureus-associated sepsis in a mammalian subjectsuitably comprise administering an effective amount of an isolatedanti-AT antibody or antigen-binding fragment thereof to the subjectprior to an infection event. As used herein, “infection event” refers toan event during which the subject is, or could be, exposed to S. aureusinfection. Exemplary infection events include, but are not limited to,surgery on any part of the body, including head, mouth, hands, arms,legs, trunk, internal organs (e.g., heart, brain, bowels, kidneys,stomach, lungs, liver, spleen, pancreas, etc.), bones, skin. Surgeryprovides conditions, such as open surgical wounds and organs, which canreadily be infected with S. aureus. Additional infection events includetrauma to any part of the body that provides open wounds or otherwiseaccess to the bloodstream via which S. aureus infection could enter thebody. Additional infection events include blood transfusions, injectionsof medications or illegal or legal drugs, needle pricks, tattoo needles,insertion and maintenance of intravenous (IV) lines, insertion andmaintenance of surgical drains, and sites of skin breakdown e.g.,bedsores (decubitus ulcers).

In embodiments where the methods provide prevention of S.aureus-associated sepsis, the anti-AT antibody or antigen-bindingfragment thereof is suitably administered at least 1 hour prior to aninfection event. For example, at least 1 hour prior to surgery (theinfection event). Suitably, the anti-AT antibody or antigen-bindingfragment thereof is administered at least 6 hours, at least 12 hours, atleast 18 hours, at least 24 hours, at least 30 hours, at least 36 hours,at least 42 hours, at least 48 hours, or longer, prior to the infectionevent. In embodiments, the anti-AT antibody or antigen-binding fragmentthereof is suitably administered about 6 hours to about 36 hours, about6 hours to about 36 hours, about 12 hours to about 36 hours, about 12hours to about 24 hours, about 24 hours to about 36 hours, about 20hours to about 30 hours, about 20 hours to about 28 hours, about 22hours to about 26 hours, or about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours,about 28 hours, about 29 hours, or about 30 hours, or about 31 hours, orabout 32 hours, or about 33 hours, or about 34 hours, or about 35 hours,or about 36 hours, prior to the infection event.

As used herein “prevention” of S. aureus-associated sepsis refers toreducing the risk of a subject acquiring S. aureus-associated sepsis atthe time of the infection event. Suitably, the risk of a subjectacquiring S. aureus-associated sepsis is reduced by at least 30% ascompared to a subject that has not been administered an anti-AT antibodyor antigen-binding fragment prior to the infection event. More suitablythe risk is reduced by at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or the risk is completelyeliminated as compared to a subject that has not been administered ananti-AT antibody or antigen-binding fragment prior to the infectionevent.

In methods for reducing the severity of S. aureus-associated sepsis in amammalian subject, such methods suitably comprise administering aneffective amount of an isolated anti-S. aureus alpha toxin (anti-AT)antibody or antigen-binding fragment thereof to a subject that isexhibiting symptoms of S. aureus-associated sepsis. Such symptoms caninclude, for example, chills, confusion or delirium, fever or low bodytemperature (hypothermia), light-headedness due to low blood pressure,rapid heartbeat, shaking, skin rash and warm skin.

As used herein “reducing the severity” as it is used with reference tosepsis refers to reducing the symptoms that a subject that has acquiredS. aureus-associated sepsis is exhibiting. Suitably, the symptoms arereduced by at least 30% as compared to the symptoms that a subject thatalso has acquired S. aureus-associated sepsis is exhibiting, but thesubject has not been administered an anti-AT antibody or antigen-bindingfragment thereof. More suitably the symptoms are is reduced by at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90% or the symptoms are completely eliminated (i.e., the subject iscured of the infection and the sepsis) as compared to a subject that hasnot been administered an anti-AT antibody or antigen-binding fragmentthereof prior to the infection event.

As used herein, the terms “treat,” “treating” or “treatment” can referto therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of thedisease. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state.“Treatment” can also mean prolonging survival as compared to expectedsurvival if not receiving treatment. Those in need of treatment includethose already with the condition or disorder as well as those prone tohave the condition or disorder or those in which the condition ordisorder is to be prevented.

Suitably subjects that can be administered the anti-AT antibodies orantigen-binding fragments thereof in the various methods describedherein are mammals, such as for example, humans, dogs, cats, primates,cattle, sheep, horses, pigs, etc.

An antibody or antigen-binding fragment thereof described herein can beadministered at a suitable dosage and dosage regimen, and such dosageand dosage regimen can depend on the disease or condition. An “effectivedosage” can be identified by determining whether a dosage and dosageregimen gives rise to a therapeutic effect or therapeutic end-point(e.g., prevention). Dosing of the antibody or antigen-binding fragmentthereof can be provided in a single administration, or over multipleadministrations spaced according to desired effects and other clinicalconsiderations.

Exemplary methods by which the anti-AT antibody or antigen-bindingfragment thereof can be administered to the subject in any of thevarious methods described herein include, but are not limited to,intravenous (IV), intratumoral (IT), intralesional (IL), aerosal,percutaneous, endoscopic, topical, intramuscular (IM), intradermal (ID),intraocular (TO), intraperitoneal (IP), transdermal (TD), intranasal(IN), intracereberal (IC), intraorgan (e.g. intrahepatic), slow releaseimplant, or subcutaneous administration, or via administration using anosmotic or mechanical pump.

In further embodiments, methods of reducing S. aureus bacterial load inthe bloodstream or heart of a mammalian subject are provided. Suchmethods suitably comprise administering to the subject an effectiveamount of an isolated anti-S. aureus alpha toxin (anti-AT) antibody orantigen-binding fragment thereof.

Bacterial load in the bloodstream or heart of a mammalian subject issuitably measured via methods known in the art to determine the amountof bacteria, suitably S. aureus bacterial colonies, in the bloodstreamor heart. For example, bacterial load is suitably measured by plating asample from an organism onto an agar plate, incubating the plate, andthen quantifying the number of colony forming units (CFU) on the plate.Such methods are well known in the art. Additional suitable methods fordetermining bacterial load can also be utilized. The collected sample issuitably from a blood sample from an organism taken generally, orspecifically, from a particular organ.

Suitably, the bacterial load (i.e., the amount of bacteria as measuredby colony forming units) of a subject infected with S. aureus is reducedby at least 30% in subjects treated with anti-AT antibodies orantigen-binding fragments thereof, as compared to subjects that alsohave been infected with S. aureus, but the subject has not beenadministered an anti-AT antibody or antigen-binding fragment thereof.More suitably the amount of bacteria is reduced by at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% or thebacterial load is completely eliminated as compared to a subject thathas not been administered an anti-AT antibody or antigen-bindingfragment.

Suitably, the anti-AT antibodies or antibody antigen-binding fragmentsthereof are administered as soon as possible after diagnosis ofinfection with S. aureus, e.g., within hours or days. The duration andamount of anti-AT antibodies or antigen-binding fragments to beadministered are readily determined by those of ordinary skill in theart.

Also provided are methods of reducing S. aureus bacterial agglutinationand/or thromboembolic lesion formation in a mammalian subject. Suchmethods suitably comprise administering to said subject an effectiveamount of an isolated anti-S. aureus alpha toxin (anti-AT) antibody orantigen-binding fragment thereof, including anti-S. aureus alpha toxin(anti-AT) antibodies or antigen-binding fragments thereof describedherein or otherwise known in the art.

As described herein, methods of reducing S. aureus bacterialagglutination refer to lowering the amount of clumping between S. aureusbacteria when in contact with blood and/or in an organ. Exemplarymethods of measuring bacterial agglutination are known in the art,including for example, as described in McAdow et al., PLos Pathogens7:e1002307 (2011), the disclosure of which is incorporated by referenceherein in its entirety. Suitably, the methods provided herein also lowerthromboembolic lesion formation in the bloodstream and/or organs of asubject. Methods of measuring thromboembolic lesion formation are knownin the art and include, for example, magnetic resonance imaging (MRI),computed tomography (CT) or computed axial tomography (CAT) scan, orother suitable imaging methods.

Methods of reducing S. aureus bacterial agglutination and/orthromboembolic lesion formation in a mammalian subject suitably resultin a reduction of bacterial agglutination and/or thromboembolic lesionformation by at least 30% in subjects treated with anti-AT antibodies orantigen-binding fragments thereof, as compared to subjects that alsohave been infected with S. aureus, but the subject has not beenadministered an anti-AT antibody or antigen-binding fragment thereof.More suitably, the bacterial agglutination and/or thromboembolic lesionformation is reduced by at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or bacterial agglutination and/orthromboembolic lesion formation is completely eliminated as compared toa subject that has not been administered an anti-AT antibody orantigen-binding fragment thereof.

Suitably, the methods of preventing S. aureus-associated sepsis in amammalian subject or reducing the severity of S. aureus-associatedsepsis in a mammalian subject also result in reduction of bacterial loadin the bloodstream or heart of the subject. In other embodiments, themethods of preventing S. aureus-associated sepsis in a mammalian subjector reducing the severity of S. aureus-associated sepsis in a mammaliansubject also result in reduction of bacterial agglutination and/orthromboembolic lesion formation in the subject.

In additional embodiments, methods of preventing or reducing theseverity of S. aureus-associated pneumonia in an immunocompromisedmammalian subject are provided. Such methods suitably compriseadministering to the subject an effective amount of an isolated anti-S.aureus alpha toxin (anti-AT) antibody or antigen-binding fragmentthereof.

As described herein, it has been surprisingly found thatimmunocompromised mammalian subjects can be administered anti-ATantibodies or antigen-binding fragments thereof so as to either preventS. aureus-associated pneumonia, or to reduce the severity of S.aureus-associated pneumonia in subjects that have already contractedpneumonia.

As used herein “immunocompromised” refers to mammalian subjects that areincapable of developing a normal immune response, and generally suchsubjects are suffering from neutropenia, which refers to an abnormallylow number of neutrophils. The severity of neutropenia is determined bythe absolute neutrophil count (ANC) measured in cells per microliter ofblood. Mild neutropenia (1000≦ANC<1500); moderate neutropenia(500≦ANC<1000); and severe neutropenia (ANC<500) are common levelsdetermined by those of ordinary skill in the art.

As used herein “prevention” of S. aureus-associated pneumonia in animmunocompromised mammalian subject refers to reducing the risk of animmunocompromised subject from acquiring S. aureus-associated pneumoniaat the time of an infection event. Suitably, the risk of animmunocompromised subject acquiring S. aureus-associated pneumonia isreduced by at least 30% as compared to an immunocompromised that has notbeen administered an anti-AT antibody or antigen-binding fragment priorto the infection event. More suitably the risk is reduced by at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90% or the risk is completely eliminated as compared to animmunocompromised subject that has not been administered an anti-ATantibody or antigen-binding fragment thereof prior to the infectionevent.

In methods for reducing the severity of S. aureus-associated pneumoniain a mammalian subject, such methods suitably comprise administering aneffective amount of an isolated anti-S. aureus alpha toxin (anti-AT)antibody or antigen-binding fragment thereof to a subject that isexhibiting symptoms of S. aureus-associated pneumonia. Such symptoms caninclude, for example, cough, chest pain, fever, and difficultybreathing.

As used herein “reducing the severity” as it is used with reference topneumonia refers to reducing the symptoms that a subject (suitably animmunocompromised subject) that has acquired S. aureus-associatedpneumonia is exhibiting. Suitably, the symptoms are reduced by at least30% as compared to the symptoms that a subject that also has acquired S.aureus-associated pneumonia is exhibiting, but the subject has not beenadministered an anti-AT antibody or antigen-binding fragment thereof.More suitably the symptoms are reduced by at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or the symptoms arecompletely eliminated (i.e., the subject is cured of the infection andtherefore pneumonia) as compared to an immunocompromised subject thathas not been administered an anti-AT antibody or antigen-bindingfragment thereof prior to the infection event.

As described herein, suitably the various methods are carried out onmammalian subject that are humans, including adults of any age andchildren.

Suitably, in the methods described herein, the antibodies orantigen-binding fragments thereof that are administered are isolated Fv,Fab, Fab′, and F(ab′)2 antigen-binding fragments. In furtherembodiments, the antibody is a full-length antibody, as describedherein. Suitably the antibody comprises an Fc variant region asdescribed in detail throughout.

The methods described throughout suitably utilize isolated antibodies orantigen-binding fragments thereof that immunospecifically bind to aStaphylococcus aureus alpha toxin polypeptide. Such antibodies andantigen-binding fragments thereof suitably comprise:

-   -   (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:        7, 10, 13 or 69;    -   (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:        8, 11, 14, 17, 70 or 75;    -   (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:        9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78;    -   (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1        or 4;    -   (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:        2, 5, 73 or 77; and    -   (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:        3, 6, 64, 68 or 74.        In addition, mutants, variants and derivatives of such        antibodies or antigen-binding fragments can also be utilized, as        well as antibodies or antigen-binding fragments thereof        exhibiting at least 90% identity to the recited amino acid        sequences.

In further embodiments, the various methods described herein utilizeantibodies or antigen-binding fragments thereof comprising CDRsincluding, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 andVL CDR3 corresponding to the amino acid sequences of SEQ ID NOs: 7, 8,9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1, 2 and 3; SEQ ID NOs: 13, 14,15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2 and 3; SEQ ID NOs: 7, 8, 16,1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 64; SEQ ID NOs; 7, 8, 66, 1,2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and 68; SEQ ID NOs: 7, 8, 67, 1, 2and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2and 68; SEQ ID NOs: 69, 70, 71, 1, 2 and 68; SEQ ID NOs: 7, 8, 72, 1, 73and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1,2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77 and 74; SEQ ID NOs: 69, 70, 71,1, 77 and 74

In embodiments, the isolated anti-AT antibody or antigen-bindingfragment thereof utilized in the various methods described hereincomprises a heavy chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45,47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and comprises a light chainvariable domain having at least 90% identity to the amino acid sequenceof SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60or 63.

In still further embodiments of the methods described herein, theisolated anti-AT antibody or antigen-binding fragment thereof utilizedcomprises a heavy chain variable domain of SEQ ID NO 20, 22, 24, 26, 28,41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and a light chainvariable domain of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50,52, 54, 56, 58, 60 or 63.

In additional embodiments, the methods described herein utilize anti-ATantibodies or antigen-binding fragments thereof have VH and VLcorresponding to the amino acid sequences of SEQ ID NOs: 20 and 19; SEQID NOs; 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ IDNOs: 28 and 27; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ IDNOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 47 and 48; SEQ IDNOs: 49 and 50; SEQ ID NOs: 51 and 52; SEQ ID NOs: 51 and 52; SEQ IDNOs: 53 and 54; SEQ ID NOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ IDNOs: 59 and 60; SEQ ID NOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ IDNOs: 62 and 63; SEQ ID NOs: 79 and 63.

In suitable embodiments, the isolated anti-AT antibody comprises anisolated anti-AT antibody having an Fc variant domain, wherein theantibody comprises a VH-IgG1-YTE corresponding to SEQ ID NO: 80 and/or aVL-Kappa corresponding to SEQ ID NO: 81.

EXAMPLES Example 1 Establishment of Sepsis Model Preparation of BacteriaChallenge Dose

S. aureus SF8300 (USA300) was provided by Binh Diep (UniversityCalifornia San Francisco). Bacteria were cultured overnight at 37° C. in50 mL of tryptic soy broth (TSB) shaking at 250 rpm. Ten mL from theovernight culture were added to 1 L of fresh TSB and the bacteria grownat 37° C. with shaking to an optical density at 600 nm (OD600) of 0.8.Bacteria were recovered by centrifugation at 8000 rpm for 15 min at 4°C. and washed in phosphate buffer saline (PBS). The bacteria werecollected by centrifugation and resuspended in PBS with 10% glycerol toa final bacterial stock concentration of ˜2×10¹⁰ cfu/mL.

Mouse Challenge and Survival

Groups of ten 8-9 week old female BALB/c mice were injectedintra-peritoneally (IP) with LC10 at indicated concentrations or R347(45 mg/kg) mAbs in 500 μL PBS. Animals were then challengedintravenously (IV) in the tail vein 24 h later with 200 μL of abacterial suspension (5×10⁷ cfu diluted in PBS, pH 7.2, from frozenstock). Mice were monitored for survival for 14 days post challenge.Statistical analysis was assessed with a log rank test: R347 (control)versus LC10 (anti-AT Ab) immunized animals.

Bacterial Load in Heart

Infected mice were euthanized with CO₂ 14 h post infection. The heartwas removed, homogenized in lysing matrix A tubes in 1 mL cold PBS, andplated on TSA plates for bacterial enumeration. The bacterial load inheart tissue was analyzed in pairwise comparison between R347 and LC10mAbs with an unpaired two-tailed Student's t-test. Data were consideredsignificant if p<0.05.

Bacteria Load in Blood

Animals were euthanized with CO₂ at 8, 24, 48, 72, and 144 h postinfection. Blood was collected by cardiac puncture, and 100 μL wasplated immediately on a TSB plate for cfu enumeration. Data wereanalyzed with an unpaired student t test. Values were consideredstatistically different between LC10 and R347 mAbs if p<0.05.

Example 2 Prophylactic Effect of Anti-AT Antibodies in Sepsis

To determine if anti-AT antibody mediated inhibition of AT would affectthe progression of sepsis, groups of 10 mice were passively immunizedwith LC10 (45 and 15 mg/kg) or an isotype control (R347, 45 mg/kg) 24-hprior to IV challenge with S. aureus SF8300 (USA300), and survivalmonitored for 14 days. LC10 prophylaxis significantly increasedsurvival, indicating AT plays a key role in systemic S. aureus diseaseand its inhibition with LC10 protects animals from death (FIG. 1).

One hallmark of S. aureus sepsis is bacterial agglutination andthromboembolic lesion formation which is measured as bacterial CFU inthe heart (McAdow et al, 2011). To determine if LC10 prophylaxis reducedthe bacterial load in the heart, mice were passively immunized with LC10(15 and 45 mg/kg) or R347 (45 mg/kg) 24 h prior to IV infection withSF8300. Fourteen-hours post infection the animals were euthanized andtheir hearts processed for CFU enumeration. Mice passively immunizedwith LC10 exhibited a significant reduction in heart CFU relative tomice that received the R347 control (FIG. 2).

The effect of LC10 prophylaxis on bacterial counts in the blood was alsoassessed 24 to 72 h post IV infection. Bacterial counts in thebloodstream of the infected mice remained at ˜10³ cfu for the R347treated mice through 72 h. However, LC10 prophylaxis resulted in reducedbacterial load at all time points tested with a maximal reduction of 2orders of magnitude at 72 h (FIG. 3). These results indicate that AT isimportant to the progression of sepsis, and inhibition of AT with LC10reduces bacterial cfu in the bloodstream and heart and promotes survivalfollowing IV challenge with a lethal dose of S. aureus.

Example 3 Establishment of an Immunocompromised Pneumonia Model

Immunocompromised individuals, particularly those suffering fromneutropenia, are at increased risk for S. aureus infections (Andrews andSullivan, 2003; Bouma et. al., 2010). To study the effectiveness ofanti-AT antibodies for use in the prevention of S. aureus pneumonia inimmunocompromised individuals, an immunocompromised murine pneumoniamodel was developed and utilized. To mimic infection in animmunocompromised population of individuals, mice were renderedneutropenic through the administration of cyclophosphamide, analkylating agent known to deplete white blood cells in mice includingneutrophils, lymphocytes and platelets (Zuluaga, et. al. 2006).

Experiments were conducted to determine the optimal cyclophosphamide(CPM) dosing regimen necessary to reduce circulating immune cells inC57BL/6 mice by >90%. CPM powder was dissolved in sterile water forinjection to a final concentration of 20 mg/mL. Groups of 20 mice weretreated by intraperitoneal injection on Days 0 and 3 with different CPMdosing regimens. Groups of 5 animals were sacrificed on Days 0, 1, 4 and6 and blood was collected by cardiac puncture into Vacutainer EDTAtubes. Differential white blood cell (WBC) counts (neutrophils,lymphocytes) were then obtained using a Sysmex automated hematologyanalyzer.

Mouse Pneumonia Model Preparation of Bacteria Challenge Dose

S. aureus SF8300 was cultured overnight at 37° C. in 50 mL tryptic soybroth (TSB) shaking at 250 rpm. Ten mL overnight culture was added to 1L fresh TSB, and the bacteria were grown at 37° C. with shaking to anoptical density of 0.8 at 600 nm (OD600). Bacteria were recovered bycentrifugation at 8000 rpm for 15 min at 4° C. and washed in phosphatebuffer saline (PBS). The bacteria were collected again by centrifugationand resuspended in PBS with 10% glycerol to a final bacteria stockconcentration of 2×10¹⁰ CFU/mL.

Immunocompromised Mouse Pneumonia Model

Initially, the minimum lethal S. aureus dose in immunocompromised micewas identified in a challenge dose titration experiment. Twenty-fourhours after the second CPM dose, the immunocompromised mice wereanesthetized with isofluorane before inoculation with 50 μL of a S.aureus suspension (1×10⁷ to 2×10⁸ CFU) into the left and right nares.Animals were placed into a cage in a supine position for recovery andobserved for lethality over a 7-day period.

Example 4 Prophylactic Effect of Anti-Staph AT Antibodies in NeutropenicPneumonia Anti-AT mAb Efficacy Study in Immunocompromised PneumoniaModel

There were 30 animals used in this experiment, randomly assigned into 3groups. The animals were administered CPM 4 days and 1 day prior toinfection. Each group was also administered either LC10 (45 or 15 mg/kg)or R347 (45 mg/kg) 24 hr prior (Day −1) to intranasal (IN) challengewith S. aureus SF8300 (5×10⁷), and survival was observed for up to 7days. Statistical significance was determined using a Log-rank test.

Verification of Immunodeficiency

Experiments were conducted to determine the optimal CPM dose regimen toreduce the total WBC count, including neutrophils, by 90%. Groups of 20mice were treated with 6 different CPM dosing regimens on Days 0 and 3.Blood samples from 5 mice in each dose group were collected on Days 0,1, 4, and 6, and total and differential WBC counts were performed usinga Sysmex hematology analyzer. On Days 4 and 6 the animals in Group 6(1st CPM dose 150 mg/kg; 2nd CPM dose 100 mg/kg [CPM150/100] exhibited a90% reduction in total WBC relative to untreated animals. There was a90% reduction in neutrophils and lymphocytes on Day 4 and day 6 in thisgroup (FIG. 4). Leukocytes began to recover at Day 7. These results areconsistent with those reported previously (Zuluaga et al., 2006).Therefore, a CPM 150/100 dose was selected to evaluate LC10 prophylaxisin immunocompromised animals.

Determination of Bacterial Challenge Dose in Immunocompromised PneumoniaModel

To determine the minimum lethal challenge dose in immunocompromisedanimals, S. aureus SF8300 was titrated (from 2×10⁸ to 1×10⁷ CFU) by INchallenge in groups of 5 mice. Lethality was observed for 7 days (FIG.5). The lowest lethal dose was 5×10⁷ CFU and was selected as the dose totest LC10 prophylaxis.

LC10 Increases Survival in Immunocompromised Pneumonia Animals

To understand the effect of LC10 prophylaxis in immunocompromised mice,CPM-injected animals were administered LC10 (45 or 15 mg/kg) or R347 at45 mg/kg 24 hours prior to IN challenge with 50 μL of a bacterialsuspension (SF8300 at 5×10⁷ CFU) and lethality was observed as describeabove.

Passive immunization with LC10 at 45 or 15 mg/kg resulted in asignificant increase in survival relative to the R347 control (p<0.0001)(FIG. 6). To confirm animals were immunocompromised in this study, bloodsamples were collected from groups of 5 uninfected mice on Days −4, −3,1, 0, 2, and 3. Total and differential WBC counts were performed, andthere was a 90% reduction in total WBC, as well as neutrophils andlymphocytes, between days −4 and −2 for these animals. The animalsexhibited severe neutropenia (≦10 neutrophils/mL blood) on Days 0-2(FIG. 7). Therefore, prophylaxis with LC10 can reduce disease severityin a S. aureus neutropenic mouse pneumonia model.

CONCLUSIONS

An S. aureus immunocompromised mouse pneumonia model was developed usingcyclophosphamide resulting in >90% reduction in circulating white bloodcells including neutrophils and lymphocytes. The mice exhibited severeneutropenia with ≦10 neutrophils/mL blood. Prophylaxis with LC10significantly improved survival in a S. aureus neutropenic mousepneumonia model. These results indicate that passive immunization withLC10 24 hours prior to S. aureus infection of mice rendered neutropenicby the administration of cyclophosphamide significantly improvessurvival. Thus, this demonstrates that anti-AT antibodies can preventdisease in immunocompromised patients.

All documents, patents, journal articles and other materials cited inthe present application are hereby incorporated by reference.

Although the present invention has been fully described in conjunctionwith several embodiments thereof with reference to the accompanyingdrawings, it is to be understood that various changes and modificationscan be apparent to those skilled in the art. Such changes andmodifications are to be understood as included within the scope of thepresent invention as defined by the appended claims, unless they departthere from.

1. A method for preventing or reducing the severity of S.aureus-associated sepsis in a mammalian subject comprising administeringto said subject an effective amount of an isolated anti-S. aureus alphatoxin (anti-AT) antibody or antigen-binding fragment thereof.
 2. Amethod of reducing S. aureus bacterial load in the bloodstream or heartof a mammalian subject comprising administering to said subject aneffective amount of an isolated anti-S. aureus alpha toxin (anti-AT)antibody or antigen-binding fragment thereof.
 3. A method of reducing S.aureus bacterial agglutination and/or thromboembolic lesion formation ina mammalian subject comprising administering to said subject aneffective amount of an isolated anti-S. aureus alpha toxin (anti-AT)antibody or antigen-binding fragment thereof.
 4. The method of claim 1,wherein S. aureus bacterial load in a bloodstream or heart of saidsubject is reduced.
 5. The method of claim 1, wherein S. aureusbacterial agglutination and/or thromboembolic lesion formation in saidsubject is reduced.
 6. A method of preventing or reducing the severityof S. aureus-associated pneumonia in an immunocompromised mammaliansubject, comprising administering to said subject an effective amount ofan isolated anti-S. aureus alpha toxin (anti-AT) antibody orantigen-binding fragment thereof.
 7. The method of claim 1, wherein saidmammalian subject is human.
 8. The method of claim 1, wherein saidisolated anti-AT antibody or antigen-binding fragment thereof isselected from the group consisting of Fv, Fab, Fab′, and F(ab′)2.
 9. Themethod of claim 1, wherein said antibody is a full-length antibody. 10.The method of claim 9, wherein said antibody comprises an Fc variantregion.
 11. The method of claim 1, wherein the isolated antibody orantigen-binding fragment thereof immunospecifically binds to aStaphylococcus aureus alpha toxin polypeptide and includes: (a) a VHCDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13 or 69;(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11,14, 17, 70 or 75; (c) a VH CDR3 comprising the amino acid sequence ofSEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78; (d) a VLCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (e) a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77;and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6,64, 68 or
 74. 12. The method of claim 11, wherein the VH CDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2 and VL CDR3 correspond to the amino acidsequences of SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1,2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 69, 70, 71, 1, 2 and68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77and 74; SEQ ID NOs: 69, 70, 71, 1, 77 and 74
 13. The method of claim 11,wherein the isolated antibody or antigen-binding fragment thereofcomprises (i) a heavy chain variable domain having at least 90% identityto the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45,47, 49, 51, 53, 55, 57, 79, 59, 61, or 62; and (ii) comprises a lightchain variable domain having at least 90% identity to the amino acidsequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54,56, 58, 60 or
 63. 14. The method of claim 11, wherein the isolatedantibody or antigen-binding fragment thereof comprises a heavy chainvariable domain of SEQ ID NO 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51,53, 55, 57, 79, 59, 61, or 62 and a light chain variable domain of SEQID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.15. The method of claim 11, wherein the VH and VL correspond to theamino acid sequences of SEQ ID NOs: 20 and 19; SEQ ID NOs; 22 and 21;SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27; SEQID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ IDNOs: 47 and 48; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; SEQ IDNOs: 51 and 52; SEQ ID NOs: 51 and 52; SEQ ID NOs: 53 and 54; SEQ IDNOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ ID NOs: 59 and 60; SEQ IDNOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ ID NOs: 62 and 63; SEQ IDNOs: 79 and
 63. 16. The method of claim 11, wherein the isolatedantibody further comprises an Fc variant domain, wherein the antibodycomprises a VH-IgG1-YTE corresponding to SEQ ID NO: 80 and/or a VL-Kappacorresponding to SEQ ID NO: 81.